ANTIBODIES AND METHODS FOR TREATMENT OF INFLUENZA A INFECTION

Abstract

The present invention provides antibodies that neutralize infection of influenza A virus. The invention also provides nucleic acids that encode and immortalized B cells and cultured plasma cells that produce such antibodies. In addition, the invention provides the use of the antibodies of the invention in prophylaxis and treatment influenza A infection.

Claims

1. An antibody comprising the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and the mutations M428L and N434S in the constant region of the heavy chain.

2. The antibody of claim 1, wherein the antibody binds to hemagglutinin of an influenza A virus.

3. The antibody of claim 1 or 2, wherein the antibody neutralizes infection with an influenza A virus.

4. The antibody of claim 3, wherein the antibody neutralizes influenza A infection at a dose, which does not exceed half of the dose required for neutralization of influenza A with a comparative antibody, which differs from said antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

5. The antibody of claim 4, wherein the dose does not exceed one third of the dose required for neutralization of influenza A with said comparative antibody.

6. The antibody of claim 4 or 5, wherein the dose does not exceed one fifth of the dose required for neutralization of influenza A with said comparative antibody.

7. The antibody of any one of the previous claims, wherein the antibody neutralizes polymorphisms HA1 P11S, HA2 D46N, and/or HA2 N49T of H3 HA; and/or polymorphism N146D of H1 HA.

8. The antibody of any one of claim 7, wherein the antibody neutralizes polymorphisms HA1 P11S, HA2 D46N, and/or HA2 N49T of H3 HA; and/or polymorphism N146D of H1 HA with IC.sub.50 fold changes of <2 relative to HA of the wild type virus.

9. The antibody of any one of the previous claims, wherein the antibody elicits a decreased anti-drug antibody response as compared to a comparative antibody differing from said antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

10. The antibody of any one of the previous claims, wherein the antibody exhibits less immunogenicity as compared to a comparative antibody differing from said antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

11. The antibody of any one of the previous claims, wherein the antibody is a human antibody.

12. The antibody of any one of the previous claims, wherein the antibody is a monoclonal antibody.

13. The antibody of any one of the previous claims, wherein the antibody is of the IgG type.

14. The antibody of claim 13, wherein the antibody is of the IgG1 type.

15. The antibody of any one of the previous claims, wherein the light chain of the antibody is a kappa light chain.

16. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 70% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

17. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 75% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

18. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 80% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

19. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 85% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

20. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 90% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

21. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence having at least 95% identity to SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

22. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence as set forth in SEQ ID NO: 8, wherein the CDR sequences as defined in claim 1 are maintained.

23. The antibody of any one of the previous claims, wherein the CH3 region of the antibody does not comprise any further mutation in addition to M428L and N434S.

24. The antibody of any one of the previous claims, wherein the Fc region of the antibody does not comprise any further mutation in addition to M428L and N434S.

25. The antibody of any one of the previous claims, wherein the antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

26. The antibody of any one of the previous claims, wherein the antibody has a heavy chain consisting of an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain consisting of an amino acid sequence as set forth in SEQ ID NO: 10.

27. The antibody of any one of the previous claims for use in prophylaxis or treatment of infection with influenza A virus.

28. The antibody for use according to claim 27, wherein the antibody is administered prophylactically.

29. The antibody for use according to claim 27 or 28, wherein the antibody is administered at a dose which does not exceed half of the dose required for prophylaxis or treatment of influenza A with a comparative antibody, which differs from said antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

30. The antibody for use according to claim 29, wherein the dose does not exceed one third of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

31. The antibody for use according to claim 29, wherein the dose does not exceed one quarter of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

32. The antibody for use according to claim 29, wherein the dose does not exceed one fifth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

33. The antibody for use according to claim 29, wherein the dose does not exceed one sixth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

34. The antibody for use according to claim 29, wherein the dose does not exceed one seventh of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

35. The antibody for use according to claim 29, wherein the dose does not exceed one eighth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

36. The antibody for use according to claim 29, wherein the dose does not exceed one ninth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

37. The antibody for use according to claim 29, wherein the dose does not exceed one tenth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

38. The antibody for use according to any one of claims 27-37, wherein the subject to be treated is at immediate risk of influenza A infection.

39. The antibody for use according to any one of claims 27-38, wherein the subject to be treated suffers from an autoimmune disease or an allergy; or is at risk of developing an autoimmune disease or an allergy.

40. A nucleic acid molecule comprising a polynucleotide encoding the antibody of any one of claims 1-26.

41. The nucleic acid molecule of claim 40, wherein the nucleic acid molecule comprises (i) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 12; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 12; and (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 13.

42. The nucleic acid molecule of claim 40 or 41, wherein the nucleic acid molecule comprises (i) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 14; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 14; and (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 15.

43. A combination of a first and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding the heavy chain of the antibody of any one of claims 1-26; and the second nucleic acid molecule comprises a polynucleotide encoding the corresponding light chain of the same antibody.

44. The combination of the first and the second nucleic acid molecule of claim 43, wherein (i) the first nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 12; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 12; and (ii) the second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 13.

45. The combination of the first and the second nucleic acid molecule of claim 43 or 44, wherein (i) the first nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 14; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 14; and (ii) the second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15; or a nucleotide sequence having 70% or more identity to SEQ ID NO: 15.

46. A vector comprising the nucleic acid molecule of any one of claims 40-42.

47. A vector comprising the combination of nucleic acid molecules of any one of claims 43-45.

48. A cell expressing the antibody of any one of claims 1-26, or comprising the vector of claim 46 or 47.

49. A pharmaceutical composition comprising the antibody of any one of claims 1-26, the nucleic acid of any one of claims 40-42, the combination of nucleic acids of any one of claims 43-45, the vector of claim 46 or 47, or the cell of claim 48, and, optionally, a pharmaceutically acceptable diluent or carrier.

50. Use of the antibody of any one of claims 1-26, the nucleic acid of any one of claims 40-42, the combination of nucleic acids of any one of claims 43-45, the vector of claim 46 or 47, the cell of claim 48 or the pharmaceutical composition of claim 49 in the manufacture of a medicament for prophylaxis, treatment or attenuation of influenza A virus infection.

51. The antibody of any one of claims 1-26, the nucleic acid of any one of claims 40-42, the combination of nucleic acids of any one of claims 43-45, the vector of claim 46 or 47, the cell of claim 48 or the pharmaceutical composition of claim 49 for use in prophylaxis or treatment of infection with influenza A virus.

52. The antibody, the nucleic acid, the combination of nucleic acids, the vector, the cell or the pharmaceutical composition for use according to claim 51, wherein the antibody, the nucleic acid, the vector, the cell or the pharmaceutical composition is administered prophylactically.

53. The antibody, the nucleic acid, the combination of nucleic acids, the vector, the cell or the pharmaceutical composition for use according to claim 51 or claim 52, wherein the antibody, the nucleic acid, the vector, the cell or the pharmaceutical composition is administered in combination with an antiviral.

54. The antibody, the nucleic acid, the combination of nucleic acids, the vector, the cell or the pharmaceutical composition for use according to claim 53, wherein the antiviral is selected from neuraminidase inhibitors and influenza polymerase inhibitors.

55. The antibody, the nucleic acid, the combination of nucleic acids, the vector, the cell or the pharmaceutical composition for use according to claim 53 or 54, wherein the antiviral is selected from oseltamivir, zanamivir and baloxavir.

56. The antibody, the nucleic acid, the combination of nucleic acids, the vector, the cell or the pharmaceutical composition for use according to any one of claims 51-55, wherein the subject to be treated suffers from an autoimmune disease or an allergy; or is at risk of developing an autoimmune disease or an allergy.

57. A combination of (i) the antibody of any one of claims 1-26, and (ii) an antiviral agent.

58. The combination of claim 57, wherein the antiviral is selected from neuraminidase inhibitors and influenza polymerase inhibitors.

59. The combination of claim 57 or 58, wherein the antiviral is selected from oseltamivir, zanamivir and baloxavir.

60. The combination of any one of claims 57-59 for use in prophylaxis or treatment of infection with influenza A virus.

61. A method of reducing influenza A virus infection, or lowering the risk of influenza A virus infection, comprising: administering to a subject in need thereof, a therapeutically effective amount of the antibody of any one of claims 1-26.

62. The method of claim 61, wherein the antibody is administered prophylactically.

63. The method of claim 61 or 62, wherein the antibody is administered at a dose which does not exceed half of the dose required for prophylaxis or treatment of influenza A with a comparative antibody, which differs from said antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

64. The method of claim 63, wherein the dose does not exceed one third of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

65. The method of claim 63, wherein the dose does not exceed one fifth of the dose required for prophylaxis or treatment of influenza A with said comparative antibody.

66. The method of any one of claims 61-65, wherein said subject is at immediate risk of influenza A infection.

67. The method of any one of claims 61-66, wherein the antibody is administered in combination with an antiviral.

68. A method of decreasing immunogenicity of an antibody comprising the heavy chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; comprising a step of introducing the mutations M428L and N434S in the constant region of the heavy chain of the antibody.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0178] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0179] FIG. 1 shows for Example 2 the plasma concentration of human antibodies FluAB_MLNS (open squares) and FluAB_wt (comparative antibody; filled circles) in macaque plasma samples assessed via ELISA until day 56.

[0180] FIG. 2 shows for Example 3 plasma concentrations of FluAB_MLNS (animals C90142, C90190) measured using an anti-CH2 antibody ELISA to quantify total human mAb or HA antigen-binding ELISA to determine functionality of the mAbs. Graphs show linear regression between total human mAb quantification and HA binding for individual animals at selected time points (days 1, 21, 56, 86, and 113).

[0181] FIG. 3 shows for Example 4 (A) the concentrations of human antibodies FluAB_MLNS and FluAB_wt in nasal swabs as measured using ELISA and normalized to urea content; and (B) Biodistribution of human antibodies FluAB_MLNS and FluAB_wt, expressed as % urea-normalized concentration in nasal swabs over plasma concentrations. Individual animal IDs and inoculated human antibody variant (FluAB_MLNS or FluAB_wt) are indicated below.

[0182] FIG. 4 shows for Example 5 the cumulative bodyweight change over time in Tg32 mice treated with either FluAB_wt (panels B, D, circles), FluAB_MLNS (panels C, E, squares) at 1 mg/kg (panels B, C, grey symbols) and 0.3 mg/kg (panels D, E, light gray symbols) or left untreated (panel A, triangles); all mice infected intranasally with PR8 virus. Individual animals are shown; The thick black line represents the average trend of BW±SD. The number of individuals per group is indicated. * p<0.05, ** p<0.01, *** p<0.001 vs control alone (A), ° p<0.05, ° ° p<0.01, all vs the relative timepoints of MEDI8852, 2-way ANOVA with Bonferroni's multiple test correction.

[0183] FIG. 5 shows for Example 5 the % of survival comparison between 1 mg/kg dose (left panel) and 0.3 mg/kg dose (right panel) in infected Tg32 male mice treated with nothing (dashed line), FluAB_wt, or FluAB_MLNS. ** p<0.01 vs untreated mice (CTR) and FluAB_MLNS 0.3 mg/kg; ° ° ° p<0.001 vs FluAB_wt, log-rank analysis, Mantel-Cox method.

[0184] FIG. 6 shows for Example 5 the circulating levels of the injected antibodies. The individual levels (μg/ml) of circulating FluAB_wt (circles) and FluAB_MLNS (squares) measured in the serum of mice, immediately before (Day 0) and 6 days after infection are shown. Bars represent the mean±SD.

[0185] FIG. 7 shows for Example 6 the plate scheme used in the in vitro neutralization assay.

[0186] FIG. 8 shows for Example 6 the neutralization activity of FluAB_MLNS and Oseltamivir alone on H1N1 (A, C) and H3N2 (B, D) virus infection.

[0187] FIG. 9 shows for Example 6 the combined neutralization activity of FluAB_MLNS and Oseltamivir on H1 (A) and H3 (B) virus infection. Data show the inhibited fraction by FluAB_MLNS alone and in combination with heteromolar concentrations of Oseltamivir both in H1N1 (A) and H3N2 (B) viral infection of MDCK cells. Data are represented as mean±SD of triplicate values, each replicate obtained in three independent culture plates.

[0188] FIG. 10 shows for Example 6 the median effect plots of combined FluAB_MLNS and Oseltamivir. The two compounds were serially diluted at the indicated constant ratios and added to MDCK cells infected with either H1 (A) and H3 (B) viral strains. The values obtained from selected combinations at non-constant ratios (NCR) are also shown.

[0189] FIG. 11 shows for Example 6 the combination indexes of FluAB_MLNS and Oseltamivir for H1N1 virus infection. Dots represent the actual experimental points at the indicated constant ratios with the cumulated drug-drug concentration denoted aside. The dotted curves show the predicted combination index across the complete effect range.

[0190] FIG. 12 shows for Example 6 the combination indexes of FluAB_MLNS and Oseltamivir for H3N2 virus infection. Dots represent the actual experimental points at the indicated constant ratios with the cumulated drug-drug concentration denoted aside. The dotted curves show the predicted combination index across the complete effect range.

[0191] FIG. 13 shows for Example 6 isobolograms of FluAB_MLNS-Oseltamivir combinations for H1N1 virus infection. Dots show the IC.sub.50, IC.sub.75 and IC.sub.90 values on different constant ratio FluAB_MLNS-Oseltamivir combinations. For each experimental point, the cumulated concentration is shown.

[0192] FIG. 14 shows for Example 6 isobolograms of FluAB_MLNS-Oseltamivir combinations for H3N2 virus infection. Dots show the IC.sub.50, IC.sub.75 and IC.sub.90 values on different constant ratio FluAB_MLNS-Oseltamivir combinations. For each experimental point, the cumulated concentration is shown.

[0193] FIG. 15 shows for Example 6 the neutralization activity of FluAB_MLNS and Zanamivir alone on H1N1 (A, C) and H3N2 (B, D) virus infection.

[0194] FIG. 16 shows for Example 6 the combined neutralization activity of FluAB_MLNS and Zanamivir on H1 (A) and H3 (B) virus infection. Data show the inhibited fraction by FluAB_MLNS alone and in combination with heteromolar concentrations of Zanamivir both in H1N1 (A) and H3N2 (B) viral infection of MDCK cells. Data are represented as mean±SD of triplicate values, each replicate obtained in three independent culture plates.

[0195] FIG. 17 shows for Example 6 the median effect plots of combined FluAB_MLNS and Zanamivir. The two compounds were serially diluted at the indicated constant ratios and added to MDCK cells infected with either H1 (A) and H3 (B) viral strains. The values obtained from selected combinations at non-constant ratios (NCR) are also shown.

[0196] FIG. 18 shows for Example 6 the combination indexes of FluAB_MLNS and Zanamivir for H1N1 virus infection. Dots represent the actual experimental points at the indicated constant ratios with the cumulated drug-drug concentration denoted aside. The dotted curves show the predicted combination index across the complete effect range.

[0197] FIG. 19 shows for Example 6 the combination indexes of FluAB_MLNS and Zanamivir for H3N2 virus infection. Dots represent the actual experimental points at the indicated constant ratios with the cumulated drug-drug concentration denoted aside. The dotted curves show the predicted combination index across the complete effect range.

[0198] FIG. 20 shows for Example 6 isobolograms of FluAB_MLNS-Zanamivir combinations for H1N1 virus infection. Dots show the IC.sub.50, IC.sub.75 and IC.sub.90 values on different constant ratio FluAB_MLNS-Zanamivir combinations. For each experimental point, the cumulated concentration is shown.

[0199] FIG. 21 shows for Example 6 isobolograms of FluAB_MLNS-Zanamivir combinations for H3N2 virus infection. Dots show the IC.sub.50, IC.sub.75 and IC.sub.90 values on different constant ratio FluAB_MLNS-Zanamivir combinations. For each experimental point, the cumulated concentration is shown.

[0200] FIG. 22 shows for Example 6 the neutralization activity of FluAB_MLNS and Baloxavir alone on H1N1 (A, C) and H3N2 (B, D) virus infection.

[0201] FIG. 23 shows for Example 6 the combined neutralization activity of FluAB_MLNS and Baloxavir on H1 (A) and H3 (B) virus infection. Data show the inhibited fraction by FluAB_MLNS alone and in combination with heteromolar concentrations of Baloxavir both in H1N1 (A) and H3N2 (B) viral infection of MDCK cells. Data are represented as mean±SD of triplicate values, each replicate obtained in three independent culture plates.

[0202] FIG. 24 shows for Example 6 the median effect plots of combined FluAB_MLNS and Baloxavir. The two compounds were serially diluted at the indicated constant ratios and added to MDCK cells infected with either H1 (A) and H3 (B) viral strains. The values obtained from selected combinations at non-constant ratios (NCR) are also plotted.

[0203] FIG. 25 shows for Example 6 the combination indexes of FluAB_MLNS and Baloxavir. Dots represent the actual experimental points at the indicated constant ratios with the cumulated drug-drug concentration denoted aside. The dotted curves show the predicted combination index across the complete effect range.

[0204] FIG. 26 shows for Example 6 isobolograms of FluAB_MLNS-Baloxavir combinations. Dots show the IC.sub.50, IC.sub.75 and IC.sub.90 values on different constant ratio FluAB_MLNS-Baloxavir combinations. For each experimental point, the cumulated concentration is shown.

[0205] FIG. 27 shows for Example 7 the binding of human FcRn in solution to immobilized FluAB_MLNS (gray line) or FluAB_wt (black line) as measured by Octet at pH=6.0 (A) or pH=7.4 (B). The time point 0 seconds represents switch from base line buffer to buffer containing human FcRn. Time point 420 seconds (gray dotted vertical line) represents switch to blank buffer at the corresponding pH. Association and dissociation profiles were measured in real time using an Octet RED96 (ForteBio).

[0206] FIG. 28 shows for Example 9 the levels of ADA response measured by ELISA to detect mouse anti-drug IgG (A; bars represent the mean±SD of treatment group); and correlation analysis (B) between the levels of circulating human IgG measured 14 days after i.v. injection (X axis) and the signal of the ADA present at the same time point (Y axis). The non-parametric Spearman's correlation coefficient is shown for the significant values.

[0207] FIG. 29 shows for Example 10 levels of ADA response after subcutaneous (s.c.) injection of either FluAB_MLNS or FluAB_wt. Data are represented as values of the ADA signal (OD 450 nm) detected in each individual serum obtained three weeks after the s.c. injection (n=5/group), pre-diluted in PBS 1:25 and then further serially diluted 5-fold.

EXAMPLES

[0208] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Safety and Tolerability of an Antibody According to the Present Invention in Cynomolgus Macagues

[0209] An antibody according to the present invention, which comprises (i) the CDR sequences as set forth in SEQ ID NOs 1-6 and (ii) the two mutations M428L and N434S in the heavy chain constant regions, was designed and produced. More specifically, the antibody comprises (i) the heavy chain variable region (VH) sequence as set forth in SEQ ID NO: 7 and the light chain variable region (VL) sequence as set forth in SEQ ID NO: 8; and (ii) the two mutations M428L and N434S in the heavy chain constant regions. Even more specifically, the antibody comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10. This antibody is referred to herein as “FluAB_MLNS”.

[0210] For comparison, antibody “FluAB_wt” was used, which differs from antibody “FluAB_MLNS” only in that it does not contain the two mutations M428L and N434S in the heavy chain constant regions. Accordingly, comparative antibody “FluAB_wt” comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10.

[0211] A single intravenous infusion of 5 mg/kg of either FluAB_MLNS or FluAB_wt in a 2.5 ml/kg volume was given in a 60-minutes intravenous infusion to three female cynomolgus macaques (Macaca fascicularis) per test group. Blood or urine for clinical chemistry and hematological analyses were collected pre-dose and on days 7 and 21 post-dose.

[0212] Following dosing of either FluAB_MLNS or FluAB_wt at 5 mg/kg in a 60-minutes intravenous infusion, the female cynomolgus macaques were closely monitored for health and weight and regularly sampled for blood and urine. No adverse events—other than bruising 24 h and erythroderma 3 days post-dose at the inoculation site in some of the animals—were observed following intravenous inoculation of the antibodies. All animals were generally healthy, showed normal food consumption, and had overall positive weight gain throughout the study. Clinical chemistry, hematology, and urinalysis parameters were normal at 7- or 21-days post dosing, compared to pre-dosing samples.

[0213] In summary, a single intravenous infusion of either FluAB_MLNS or FluAB_wt into cynomolgus macaques did not induce adverse events and was generally well tolerated.

Example 2: Determination of Plasma Concentration and Pharmacokinetics

[0214] These experiments aimed to determine the concentration, establish half live, and compare the pharmacokinetics of the antibody according to the present invention FluAB_MLNS in comparison to comparative antibody FluAB_wt in the plasma following a single intravenous injection.

[0215] Before dosing, the animals were tested to be negative for influenza-specific antibodies using dot immunobinding assay. Seropositive animals were excluded from the study as pre-existing immunity may interfere with this test. In addition, animals developing anti-drug antibody (ADA) response were excluded.

[0216] A single intravenous infusion of 5 mg/kg of either FluAB_MLNS or FluAB_wt in a 2.5 ml/kg volume was given in a 60-minutes intravenous infusion to three female macaques per test group. Blood was collected in tubes containing K.sub.2EDTA pre-dose and processed to plasma for pharmacokinetic testing after approximately 1, 6, 24, 96, 168, 504, 840, and 1344 hours (h) post-dose.

[0217] Plasma concentration of the antibodies was determined in vitro using an ELISA assay. Briefly, IAV-HA antigen (Influenza A virus H1N1 A/California/07/2009 Hemagglutinin Protein Antigen (with His Tag); Sino Biologicals) was diluted to 2 μg/ml in PBS and 25 μl were added to the wells of a 96-well flat bottom %-area ELISA plate for coating over night at 4° C. After coating, the plates were washed twice with 0.5×PBS supplemented with 0.05% Tween20 (wash solution) using an automated ELISA washer. Then, plates were blocked with 100 μl/well of PBS supplemented with 1% BSA (blocking solution) for 1 h at room temperature (RT) and then washed twice. Plasma samples were centrifuged at 10′000 g for 10 min at 4° C. and then diluted (1:10 and then 1:30) for a final 1:300 dilution in blocking solution in 96-well cell culture plates. The minimum dilution (1:300) of the macaque plasma used for quantification was tested and set to ensure that the matrix effect was negligible. Samples were then diluted 1:2 stepwise in triplicates for a total of 12 dilutions. Standards for each antibody to be tested were prepared similarly via diluting the antibodies 1:300 to 1 μg/ml in a pool of pre-inoculation plasma from all test animals, mimicking the matrix of the test samples. Standards were then diluted 1:3 stepwise in blocking solution in triplicates for a total of 12 dilutions. Twenty-five μl of the prepared samples or standards were added to hemagglutinin (HA)-coated wells and incubated for 1 h at RT. After four washes, 25 μl of goat anti human-IgG HRP conjugate (AffiniPure F(ab′).sub.2 Fragment, Fcγ Fragment-Specific; Jackson ImmunoResearch) diluted in blocking solution 1:5′000 (final concentration 0.16 μg/ml) were added per well for detection and incubated at RT for 1 h. After four washes, plates were developed by adding 40 μl per well of SureBlue TMB Substrate (Bioconcept). After ˜7-20 min incubation at RT, when the color reaction reached a plateau (max OD˜3.8), 40 μl of 1% HCl were added per well to stop the reaction and absorbance was measured at 450 nm using a spectrophotometer.

[0218] To determine the concentration of the antibodies in cynomolgus plasma, OD values from ELISA data were plotted vs. concentration in the Gen5 software (BioTek). A non-linear curve fit was applied using a variable slope model, four parameters and the equation: Y=(A−D)/(1+(X/C){circumflex over ( )}B)+D).

[0219] The OD values of the sample dilutions that fell within the predictable assay range of the standard curve as determined in setup experiment by quality control samples in the upper, medium or lower range of the curve were interpolated to quantify the samples. Plasma concentration of the antibodies were then determined considering the final dilution of the sample. If more than one value of the sample dilutions fell within the linear range of the standard curve, an average of these values was used. Pharmacokinetics (PK) data were analyzed by using WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (8.1.0.3530 Core Version, Phoenix software, Certara) with the following settings: Model: Plasma Data, Constant Infusion Administration; Number of non-missing observations: 8; Steady state interval Tau: 1.00; Dose time: 0.00; Dose amount: 5.00 mg/kg; Length of Infusion: 0.04 days; Calculation method: Linear Trapezoidal with Linear Interpolation; Weighting for lambda_z calculations: Uniform weighting; Lambda_z method: Find best fit for lambda_z, Log regression. Graphing and statistical analyses (linear regression or outlier analysis) were performed using Prism 7.0 software (GraphPad, La Jolla, Calif., USA). Outlier analysis was performed using the ROUT method (Q=1%), with the potential to find any number of outliers in either direction.

[0220] Results are shown in FIG. 1. Analysis of cynomolgus plasma samples drawn up to 56 days post-inoculation demonstrated that the antibody according to the present invention FluAB_MLNS had an extended in-vivo half-live compared to comparative antibody FluAB_wt (FIG. 1). Using noncompartmental analysis with WinNonLin, the T.sub.1/2 was estimated as 19.5 days for the antibody according to the present invention FluAB_MLNS, while T.sub.1/2 was estimated as 11.6 days for the comparative antibody FluAB_wt. The lower limit of quantification was 300 ng/ml.

[0221] In summary, the antibody according to the present invention FluAB_MLNS had an extended in-vivo half-live compared to comparative antibody FluAB_wt at least up to day 56 post-inoculation.

Example 3: Long-Term Stability In Vivo

[0222] To test in-vivo stability and functionality of the antigen binding of the antibody according to the present invention FluAB_MLNS over time, the pharmacokinetics measurement (as described in Example 2) of the group receiving the antibody according to the present invention FluAB_MLNS was extended to days 86 and 113 post-inoculation. On days 1, 21, 56, 86, 113 post-inoculation, functional FluAB_MLNS was quantified using the hemagglutinin (HA) binding ELISA as described in Example 2.

[0223] Further, total human antibodies in macaque plasma was quantified using a specific anti-CH2 ELISA, using a capture mAb that specifically binds the CH2 region of human but not of monkey Abs. To measure total human IgG and thus quantify total inoculated human antibodies in cynomolgus plasma, an ELISA capturing with mouse anti-CH2 domain-specific to human IgG (clone R10Z8E9; Thermo Scientific) was used. It was verified that this mAb does not cross-react with monkey IgG. For coating of 96-well flat bottom %-area ELISA plates, mouse anti-human IgG CH2 was added in PBS at 0.5 μg/ml and incubated over night at 4° C. Then, plates were washed and 100 μl/well blocking solution with 5% BSA was added for 1 h at RT. Standards of the antibody according to the present invention FluAB_MLNS were prepared via diluting the FluAB_MLNS to 1 ng/ml in blocking solution. Standards were then diluted 1:1.5 stepwise in blocking solution in duplicates for a total of 12 dilutions. Cynomolgus plasma samples were centrifuged at 10,000 g for 10 min at 4° C. and step-wise diluted to a final 1:1,000, 1:5,000 or 1:15,000 in blocking solution. After washing the plate, 25 μl of samples or standard were added to the ELISA plate and incubated for 1 h at RT. After three washes, 25 μl of goat anti human-IgG HRP (AffiniPure F(ab′).sub.2 Fragment, Fcγ Fragment-Specific; Jackson ImmunoResearch) at 0.04 μg/ml were added in blocking solution with 1% BSA for detection and incubated at RT for 45 min. After three washes, plates were developed by adding 40 μl per well of SureBlue TMB Substrate (Bioconcept). After 20 min incubation at RT, 40 μl of 1% HCl were added to stop the reaction, and absorbance was measured at 450 nm.

[0224] Results are shown in FIG. 2. Both quantifications resulted in similar human antibody concentrations in cynomolgus plasms (FIG. 2). Additional analysis via linear regression demonstrated that the relation between quantification via HA binding and total anti-CH2 quantification followed a linear patter for all selected time points. Consequently, the total amount of FluAB_MLNS present in plasma was functional in binding to the hemagglutinin (HA) stem region of influenza A virus (IAV), also after 86 and 113 days in vivo.

[0225] In summary, the antibody according to the present invention FluAB_MLNS demonstrated functional antigen binding and thus good long-term stability in vivo up to day 113 post-inoculation during study extension.

Example 4: Antibody Concentration in Nasal Swabs and Biodistribution

[0226] To determine biodistribution of the antibody according to the present invention FluAB_MLNS and of the comparative antibody FluAB_wt between the nasal mucus relative to plasma, the concentration of the antibody was determined in nasal swabs. To this end, Nasal swabs of the macaques described in Example 2 were collected 24, 504, and 1344 hours after administration of the antibody according to the present invention FluAB_MLNS or of the comparative antibody FluAB_wt. Concentrations of antibodies FluAB_MLNS and FluAB_wt in nasal swabs were determined essentially as described in Example 2 for determination in plasma with the following minor adaptations: (a) ELISA plates were blocked 2 h at RT; (b) Nasal swab samples were diluted starting at 1:2 with 1% BSA in PBS and then serially diluted step-wise 1:2 for a total of 8 dilution points; (c) nasal swab medium (RT MINI Viral Transport Medium; Copan) was used as assay matrix control.

[0227] To eliminate differences during the swabbing procedure or in the amount of nasal secretions present in each animal and at different time points (days 1, 21, and 56), results from nasal swabs were normalized to urea content. Urea freely diffuses between blood, being present in similar amounts across these plasma or swab samples (Lim et al., 2017, Antimicrob Agents Chemother 61(8):e00279-17). To this end, Urea Nitrogen (BUN) was measured quantitatively using the “Urea Nitrogen (BUN) Colorimetric Detection Kit” (Invitrogen), following the manufacture's procedure. In brief, samples were diluted 1:3 in PBS and mixed with the kit reagents A and B and incubated at room temperature for 30 minutes. The colored product of the redox reaction was read at 450 nm using a 96-well microplate reader. Quantification was performed via comparing samples to BUN standards, which were provided with the kit and treated equivalently.

[0228] Results are shown in FIG. 3. Amounts of normalized antibodies in nasal swabs decreased over time (FIG. 3A). Determining biodistribution via comparing nasal to plasma concentrations revealed no differences between the antibody according to the present invention FluAB_MLNS and the comparative antibody FluAB_wt (FIG. 3B), suggesting that the MLNS-Fc mutation, while prolonging the half-life of FluAB_MLNS in plasma, did not enhance bio-distribution of the antibody into the nasal mucus.

[0229] In summary, nasal swab samples did not reveal any significant differences in biodistribution between the nasal mucus and plasma amongst the three mAb variants.

Example 5: Prophylactic Activity of Antibody FluAB_MLNS in PR8-Infected Tg32 Mice

[0230] Next, the prophylactic activity of the antibody according to the present invention FluAB_MLNS compared to antibody FluAB_wt was determined in a H1N1 murine model of lethal influenza A infection.

[0231] To evaluate the prophylactic efficacy, 9- to 14-week-old FcRn−/− hFcRn line 32 Tg mice (C57B6 background) were intravenously (i.v.)-injected (via the tail vein) with 5 ml/kg of a solution containing the antibody according to the present invention FluAB_MLNS or the comparative antibody FluAB_wt at doses ranging from 0.3 to 1 mg/kg. Twenty-four hours after the i.v. injection, mice were bled from the tail vein to determine the serum antibody levels before infection. Bleedings were also repeated on day 6 and 13 post infection (p.i.). Both antibody-injected and untreated mice were anaesthetized (isoflurane, 4% in O.sub.2, 0.3 L/min) and challenged intranasally (i.n.) by slow instillation in both nostrils of 50 μl (25 al/each) of PBS containing 5 mouse lethal dose fifty percent (5 MLD.sub.50, equivalent to 1200 TCID.sub.50/mouse) of influenza virus A (H1N1, A/Puerto Rico/8/34, as described in Cottey, R., Rowe, C. A., and Bender, B. S. (2001). Influenza virus. Curr Protoc Immunol Chapter 19, Unit 19.11-19.11.32). Each mouse was held upright with its head tilted slightly back for about 1 minute to reduce the likelihood of inoculum dripping from the nares. After the procedure and upon righting reflex occurrence, animals were returned to the cage. The mice were monitored daily for weight loss and disease symptoms until day 14 p.i. and euthanized if they lost more than 20% of their initial body weight (whereby 0% is set on the day of infection) or reached morbidity score of 4. Table 1 details the applied morbidity score:

TABLE-US-00001 TABLE 1 Morbidity Score of PR8-infected mice Morbidity Score Clinical signs 1 Healthy 2 Consistently ruffled fur on the neck 3 Piloerection, possible deeper breathing, less alert 4 Labored breathing, tremors and lethargy 5 Abnormal gait, reduced mobility, emaciation, tail-ears cyanosis 6 Death

[0232] All the animals were eventually sacrificed to collect serum and lungs.

[0233] Serum Preparation:

[0234] Approximately 0.05 ml of blood were collected into gel-containing tubes and let stay for 30 min at RT. Tubes were spun for 5 min at 5500 rpm (3200×g), serum was transferred to new tubes and stored at −20° C. until use.

[0235] Two independent experiments were carried out, according to the following designs:

TABLE-US-00002 TABLE 2 Study Design Experiment 1: Group N of animals IV Treatment mAb Dose 1 4 — — 2 8 FluAB_wt 1 mg/kg 3 4 FluAB_wt 0.3 mg/kg 4 8 FluAB_MLNS 1 mg/kg 5 4 FluAB_MLNS 0.3 mg/kg

TABLE-US-00003 TABLE 3 Study Design Experiment 2: Group N of animals IV Treatment mAb Dose 1 9 — — 2 10  FluAB_wt 0.3 mg/kg 3 6 FluAB_MLNS 0.3 mg/kg

[0236] ELISA Quantification of Circulating mAb:

[0237] Sera were assessed for the levels of circulating antibodies on day 0 and 6. Briefly, half-area ELISA plates were coated over night at 4° C. with recombinant hemagglutinin (HA) from H1N1 strain A/California/07/09 (2 g/ml, in PBS, 25 μl/well). Following blocking (PBS/1% BSA, 100 μl/well, 1 hr RT) and 2 washes (220 /well) with ELISA washing solution (PBST), both dilutions of the sera (initial dilution 1:150 for 1 mg/kg, 1:50 for 0.3 mg/kg) and the antibody standards (FluAB_MLNS and FluAB_wt, 0.1 g/ml) were added (25 /well) in duplicate and serially diluted (1:2 by 10 points for serum dilutions, 1:3 by 8 points for antibody standards). After 1.5 hr RT incubation, plates were washed 4 times with PBST and further incubated 1.5 hr at RT with the HRP-labeled anti-human secondary antibody (0.16 Mg/ml, 25 DI/well). After 4 washes with PBST, plates were dispensed with substrate solution (25 DI/well), developed for 14 min and blocked with 1% HCl (v/v, 25 DI/well). Plates were finally read at 450 nm with a spectrophotometer for signal quantification. Concentration values were calculated by using a non-linear regression model (variable slope model, four parameters, GraphPad Prism) of log (agonist) versus response.

[0238] Data Analysis:

[0239] Data were plotted and analyzed using GraphPad Prism software version 8.0 for Macintosh, GraphPad Software, La Jolla Calif. USA, www.graphpad.com. Continuous variables were assessed for statistically significant difference (p<0.05, 95% confidence interval) by using ordinary 2-way ANOVA corrected with Bonferroni multiple comparison test. Survival data were compared by using log-rank analysis with Mantel-Cox method (p<0.05 considered statistically significant). The data from the two independent experiments described above were pooled.

[0240] Results:

[0241] The prophylactic activity was tested upon i.v. administration of FluAB_MLNS and FluAB_MLNS (1 and 0.3 mg/kg) in Tg32 mice one day prior to H1N1 PR8 virus challenge via intranasal infection. Results are shown in FIGS. 4-6.

[0242] As depicted in FIG. 4, mice treated with either 1 mg/kg (panel D) or 0.3 mg/kg (panel E) of FluAB_MLNS showed lower body weight loss, in comparison with both untreated (panel A) and FluAB_wt-injected (panels B and C) mice.

[0243] The better protective activity of FluAB_MLNS as compared to FluAB_wt was confirmed in the survival analysis shown in FIG. 5.

[0244] The differences in the efficacy between FluAB_MLNS and FluAB_wt did not correlate with different levels of circulating antibodies in the serum, as measured 1 and 7 days after i.v. administration of the antibodies (FIG. 6). Of note, no detectable levels of circulating antibodies were measured 14 days after injection (not shown).

[0245] In summary, FluAB_MLNS demonstrated, in Tg32 mice, a better protective capacity against H1N1 PR8 intranasal virus challenge over the comparative antibody FluAB_wt. The efficacy was independent of the circulating antibody levels. These data suggest that the enhanced interaction of FluAB_MLNS with hFcRn expressed by Tg32 mice also mediates in vivo effects unrelated to the extended antibody half-life, such as increased efficacy regarding the protective activity.

Example 6: Combination of Antibody FluAB_MLNS with Various Antivirals

[0246] Drug combinations offer the clear opportunity to enhance the potency while reducing the probability to select resistances. Moreover, a putative additive or synergic effect may end up to a dose-sparing approach. Influenza medications currently approved by FDA include the neuraminidase inhibitors oseltamivir and zanamivir as well as the recently approved baloxavir marboxil, which belongs to the endonuclease inhibitors class.

[0247] To evaluate the combined activity of the antibody of the invention FluAB_MLNS with the antivirals oseltamivir, zanamivir or baloxavir marboxil on both H1N1 and H3N2 representative viral strains, in vitro neutralization was performed to evaluate the resulting inhibitory effect. The analysis of the combined effects was carried out by using the median-effect plot and the calculation of the combination index (CI).

[0248] Briefly, MDCK (Madin-Darby canine kidney) cells were seeded at 30,000 cells/well into 96-well plates (flat bottom, black). Cells were cultured at 37° C. 5% CO.sub.2 overnight. Twenty-four hours later, 4× antibody and antiviral (oseltamivir, zanamivir or baloxavir marboxil) dilutions in 60 μl infection medium (MEM (Sigma Aldrich, cat. n. M0644)+Glutamax (Invitrogen, 41090-028)+10 g/ml TPCK-treated Trypsin (Worthington Biochemical #LS003750)+10 g/ml Kanamycin) were prepared by using crisscross 1:2 serial dilutions of FluAB_MLNS (starting from 166.7 nM final, 9 horizontal points) and different antivirals (oseltamivir, zanamivir or baloxavir marboxil), starting from 125 (250 for zanamivir) nM by 7 vertical points), according to the plate scheme shown in FIG. 7.

[0249] For each combination, three independent plates were prepared, in order to have triplicates of each drug-drug combination ratio. The single compound titration (namely, FluAB_MLNS, 9 points and each antiviral, 8 points) was included in each plate. Virus solution was prepared at concentrations of 120× the TCID50 in 60 μl, further diluted either 1:1 in MEM or mixed 1:1 with FluAB_MLNS dilutions and incubated 1 h at 33° C. Cells were washed 2 times using 200 μl/well MEM without supplements, followed by the addition of either 100 μl of virus alone or 100 μl of FluAB_MLNS/virus mix (100×TCID50/well) and incubated 4 hours at 33° C. 5% CO2. After the addition of 100 μl/well of infection medium, cells were further incubated for 72 hours at 33° C. 5% C02. On day 3 after infection, 20 μM MuNANA (4-MUNANA (2_-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich) #69587) solution was prepared in MuNANA buffer (MES 32.5 mM/CaCl.sub.2 4 mM, pH 6.5) and 50 μl/well was dispensed into black 96-well plates. Fifty μl of either neutralization or virus-alone titration supernatant were transferred to the plates and incubated 60 min at 37° C. The reaction was then stopped with 100 μl/well 0.2 M glycine/50% EtOH, pH 10.7. Fluorescence was quantified at 460 nm with a fluorimeter (Bio-Tek).

[0250] The fraction of virus neutralization was calculated according to the formula:

[00001]$1-\left(\frac{fx-f\min }{f\max }\right),$

[0251] wherein fx=sample fluorescence signal (cells+virus+FluAB_MLNS+antiviral); fmin=minimal fluorescence signal (cells alone, no virus); fmax=maximal fluorescence signal (cells+virus only).

[0252] The neutralized fraction data were used to compute the quantitative analysis of dose-effect relationships for drug-drug combinations according to the Chou and Talalay method (Chou T C, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22:27-55). The combination Index, the fraction affected (Fa), and isobolograms were obtained by using the CompuSyn software (ComboSyn Inc., Paramus, N.J., USA) (Chou T-C: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 2006, 58:621-681).

[0253] Results are shown in FIGS. 8-26 and described below.

[0254] Combination of FluAB_MLNS and Oseltamivir

[0255] The relative efficacy of FluAB_MLNS and oseltamivir to neutralize influenza A viruses was compared in vitro on two viral serotype representatives for both H3N2 and H1N1 strains. As shown in FIG. 8, both compounds, tested separately, were dose-dependently capable to fully inhibit cell infection, when independently exposed together with H3N3 and H1N1 virus (FIG. 8A,B). The IC.sub.50 values, as calculated from the median-effect plot (FIG. 8C,D) after data log linearization (as described in (Chou T C, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22:27-55) are indeed in the nanomolar range for both FluAB_MLNS (17.9 and 15.6 nM for H3 and H1 strains respectively) and oseltamivir (7 and 9.1 nM for H3 and H1 strains respectively). Overall, no substantial differences were measured in terms of inhibitory response by FluAB_MLNS between H3 and H1 virus infection, while H1N1 virus resulted marginally more sensitive to the inhibitory effect of oseltamivir.

[0256] To test the effect of a combination of FluAB_MLNS and oseltamivir in neutralizing the infection of MDCK cells with H3 and H1 virus, both compounds were serially diluted at different ratios as described above, and assessed for the enzymatic activity of neuraminidase (NA; as a read out of the viral content in the culture) in the presence of the different drug concentrations and compared to the single drug effects. The neutralization effect measured with FluAB_MLNS is greatly enhanced by the concomitant presence of heteromolar concentrations of the second compound, thus suggesting a synergistic effect rather than an addictive one, both on H3 and H1 virus infection (FIG. 9). A slightly different susceptibility of H1 and H3 viruses to the inhibitory action of oseltamivir was detected.

[0257] To precisely quantify the putative synergistic effects of the various drug combination ratios, the neutralization data were further transformed according to the median-effect principle and analyzed with the CompuSyn software as described above. The effects of several different FluAB_MLNS-oseltamivir combination constant ratios were plotted in the median-effect plot as shown in FIG. 10.

[0258] The CompuSyn software applies the logarithmic transformation of the median-effect equation to the experimental data and calculates both the potency (IC.sub.50) and the so-called combination index (CI) of the various drug combinations. The CI is a Chou-Talalay (median-effect) equation-derived parameter that considers the physico-chemical properties of the mass-action law and results from the sum of the two ratios between the portion of the dose of drug 1 combined with drug 2 to achieve a certain effect divided by dose of the single drug 1 and 2 to obtain the same effect. According to this mathematical algorithm, a CI=1 indicates an addictive effect, CI<1 indicates synergism and CI>1 indicates antagonism.

[0259] As shown in FIGS. 11 and 12, for all the combination ratios tested and both for H1 (FIG. 11) and H3 virus (FIG. 12), the predicted CI values across the inhibited fraction range described a curve well below 1 for all drug combination ratios and the actual experimental points of the different combined concentrations also ranged below 1 for nearly all combinations. Altogether, the data indicate a straightforward synergistic effect of FluAB_MLNS and oseltamivir when combined.

[0260] The same data can be alternatively described with isobolograms plots, which compare the equipotent concentrations of both the single and combined drugs. As shown in FIGS. 13 and 14, the distribution of the IC.sub.50, IC.sub.75, and IC.sub.90 values for the three different combination ratios is by far below the isobole lines connecting the respective IC.sub.50, IC.sub.75, and IC.sub.90 of the single drugs tested, both for H1 (FIG. 13) and H3 (FIG. 14), indicating consistent synergy (while an additive and antagonism would generate equipotency points localized either onto or over the single-drug isobole, respectively).

[0261] Combination of FluAB_MLNS and Zanamivir

[0262] The relative efficacy of FluAB_MLNS and zanamivir to neutralize influenza A viruses was also compared in vitro on two viral serotype representatives for both H3N2 and H1N1 strains. As shown in FIG. 15, both compounds, tested separately, were dose-dependently capable to fully inhibit cell infection, when independently exposed together with H3N3 and H1N1 virus. The relative calculated IC.sub.50 values were 23.1-24.4 nM for FluAB_MLNS and 10.7-13.7 nM for zanamivir.

[0263] For the combined effect of FluAB_MLNS and zanamivir FIG. 16 shows that, similarly to oseltamivir, zanamivir greatly enhances the inhibitory capacity of FluAB_MLNS both against H1 and H3 viruses.

[0264] The quantification of the synergic effect was similarly computed with CompuSyn and the median effect principle as described above. The median effect plots for the combined effects of FluAB_MLNS and zanamivir are shown in FIG. 17. The calculated CI for FluAB_MLNS and zanamivir is shown in FIGS. 18 and 19 and clearly indicates a synergistic effect between the two drugs, both with H1 (FIG. 18) and H3 (FIG. 19) viruses, as indicated by the values lower than 1 for all the experimental point tested. Consistently, with both viral strains, the isobolograms denote a strong synergistic effect across the IC.sub.50, IC.sub.75, and IC.sub.90 values (shown in FIGS. 20 and 21), which are all significantly below the IC values with the single drug.

[0265] Combination of FluAB_MLNS and Baloxavir Marboxil

[0266] The recently approved endonuclease inhibitor baloxavir marboxil was initially compared with FluAB_MLNS alone on both H1 and H3 strains, similarly as described above for oseltamivir and zanamivir. Results are shown in FIG. 22. The relative calculated IC.sub.50 values were 20.1-15.4 nM for FluAB_MLNS and 4.9-2.3 nM for baloxavir marboxil.

[0267] Although Baloxavir has a different mechanism of action in inhibiting viral replication compared to the NA inhibitors, the drug is still able to strongly enhance the inhibitory capacity of FluAB_MLNS, clearly indicating a synergistic effect (FIG. 23). The inhibition data obtained with the different combination ratios were used to compute and plot the median effect with CompuSyn software and calculate the type of drug-drug interaction as described above (FIG. 24). The calculated CI for FluAB_MLNS and baloxavir marboxil (FIG. 25) clearly indicate a synergistic effect between the two drugs, both with H1 and H3 viruses, as indicated by the values lower than 1 for the majority of the experimental points tested. The isobolograms denote a robust and complete synergistic effect across the IC.sub.50, IC.sub.75, and IC.sub.90 values (FIG. 26).

[0268] In summary, the neutralization capacity of FluAB_MLNS against both H1 and H3 strains is synergistically enhanced by different antivirals, namely, the NA inhibitors oseltamivir and zanamivir as well as the endonuclease inhibitor baloxavir-marboxil.

Example 7: Binding to Human FcRn at Different pHs

[0269] FluAB_wt and FluAB_MLNS were compared side by side for their ability to bind to neonatal Fc receptor (FcRn) using biolayer interferometry (BLI).

[0270] To this end, binding of FluAB_wt and FluAB_MLNS to human FcRn was measured on an Octet RED96 instrument (biolayer interferometry, BLI, ForteBio). Biosensors coated with anti-human Fab-CH1 were pre-hydrated in kinetic buffer for 10 min at RT. Then, human mAb (FluAB_wt or FluAB_MLNS) was loaded at 1 μg/ml in kinetics buffer at pH 7.4 for 30 minutes onto the Biosensors. The baseline was measured in kinetics buffer (Sterile filtered 0.01% endotoxin-free bovine serum albumin, 0.002% Tween-20 (Polysorbate 20), 0.005% NaN3 in PBS) at pH=7.4 or pH=6.0 for 4 minutes. Human mAb-loaded sensors were then exposed for 7 minutes to a solution of human FcRn at 1 μg/ml in kinetics buffer at pH=7.4 or pH=6.0 to measure association of FcRn-mAb in different milieus (on rate). Dissociation was then measured in kinetics buffer at the same pH for additional 5 minutes (off rate). All steps were performed while stirring at 1000 rpm at 30° C. Association and dissociation profiles were measured in real time as change in the interference patterns.

[0271] As shown in FIG. 27, FluAB_MLNS bound human FcRn with higher affinity compared to FluAB_wt at acidic pH (pH 6.0), while neither FluAB_MLNS nor FluAB_wt binds FcRn at neutral pH (pH 7.4).

Example 8: Characterization of Polymorphisms Identified in the Antibody's Extended Epitope

[0272] Historical polymorphisms in the extended epitope were evaluated for their impact on neutralization activity of FluAB_MLNS using viruses generated by reverse genetics with H1 HA or H3 HA on a A/Puerto Rico/8/34 (PR8) background.

[0273] Single nucleotide polymorphisms were introduced into PR8 H1 HA or A/Aichi/2/68 (Aichi) HA pHW2000 plasmids using site-directed mutagenesis. Recombinant influenza A virus were rescued with associated H1 or H3 HA on a PR8 backbone using standard methods (e.g., as described in Erich Hoffmann, Gabriele Neumann, Yoshihiro Kawaoka, Gerd Hobom, Robert G. Webster, 2000, A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences May 2000, 97 (11): 6108-6113; doi: 101073/pnas.100133697).

[0274] Neutralization activity was evaluated in MDCK cells using standard methods. For example, neutralization activity may be evaluated in MDCK cells, e.g. in 96 well plates. To this end, MCDK cells may be seeded at 30,000 cells/well 24 hours prior to infection. Antibody FluAB_MLNS may be incubated with virus for 1 hour at 37° C. prior to addition to MDCK cells. To this end, 1:2.5 9-point serial dilutions of FluAB_MLNS may be created in infection media and each dilution may be tested in triplicate (e.g., 50 μg/mL-0.03 μg/mL final concentration) and may be incubated with 120 TCID.sub.50 of virus for 1 hour at 37° C. MDCK cells may be washed twice with PBS, 100 μl/well of virus:antibody solution may be added, and cells may be incubated for 4 hours at 37° C. After 4 hours, an additional 100 μl/well of infection media may be added to cells. After 72 hours of incubation at 37° C., viral RNA may be extracted and measured by qRT-PCR, e.g. using WHO primers (World Health Organization. CDC protocol of real-time RT-PCR for influenza A H1N1. Apr. 28, 2009). The IC50 is expressed as the antibody concentration in μg/mL that reduces 50% of virus replication and may be calculated using a non-linear 4-parameter logistic fit curve of data normalized to control wells (no virus and virus alone).

[0275] The neutralization activity of FluAB_MLNS to H1 and H3 HA polymorphisms in the extended epitope is shown in Table 4 below.

TABLE-US-00004 TABLE 4 Amino FluAB_MLNS Acid Geomean Fold change Changes Neutralization relative to Virus in HA IC.sub.50 (μg/mL) WT virus PR8: Aichi HA wt wild type 5.6 NA PR8: Aichi HA P11S P11S 9.5 1.7 PR8: Aichi HA D46N D46N 3.3 0.6 PR8: Aichi HA N49T N49T 5.0 0.9 PR8 wt wild type 4.7 NA PR8 HA N146D N146D 5.5 1.2 Aichi = A/Aichi/2/68; Geomean = geometric mean; HA = hemagglutinin; NA = not applicable; PR8 = A/Puerto Rico/8/34 H1N1; wt = wild type

[0276] For viruses encoding H3 HA, FluAB_MLNS neutralized viruses with mutations HA1 P11S, HA2 D46N, or HA2 N49T with IC.sub.50 values similar to wild type virus (<2-fold change in IC.sub.50 relative to wild type virus). For viruses encoding H1 HA, FluAB_MLNS neutralized viruses with encoding HA2 N146D with IC.sub.50 values similar to wild type virus (<2-fold change in IC.sub.50 relative to wild type virus). Additionally, the PR8 wild type strain used encoded the HA2 polymorphism L38Q and D46N and was neutralized with an IC.sub.50 value of 4.7 μg/mL by FluAB_MLNS. Overall, all polymorphisms evaluated resulted in IC.sub.50 fold changes of <2 relative to the wild type virus for FluAB_MLNS. In summary, FluAB_MLNS effectively neutralized all evaluated historical polymorphisms in the extended epitope (H3 HA: HA1 P11S, HA2 D46N, or HA2 N49T; H1 HA: N146D).

Example 9: Anti-Drug Antibody Response in Tg32 Mice

[0277] With regard to the M428L/N434S mutation, recently concerns were raised that said mutation increases immunogenicity of antibodies comprising this mutation (Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina Radošević & Huawei Qiu (2019) Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life, mAbs, 11:7, 1276-1288; Maeda A, Iwayanagi Y, Haraya K, et al. Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody. MAbs. 2017; 9(5):844-853).

[0278] To assess immunogenicity, in particular the anti-drug response (anti-drug antibodies; ADA), of antibody FluAB_MLNS in comparison to its parental antibody FluAB_wt, two separate groups (n=5) of TG32 mice (transgenic for the human FcRn) were injected i.v. with 5 mg/kg of either FluAB-MLNS or FluAB_wt monoclonal antibodies. To evaluate the circulating levels of the injected mAbs, blood samples were then obtained at different time points. Samples taken at day 14 and 21 post injection were used to evaluate, by specific ELISA, the anti-drug antibody (ADA) response against the injected human monoclonals.

[0279] Briefly, purified FluAB_wt and FluAB_MLNS monoclonal antibodies were coated on 96-well plates at 2 μg/ml. After blocking, the sera from treated animals obtained 14 and 21 days post injection, diluted 1:180, were incubated 1.5 h at room temperature (RT). After washings, peroxidase-labeled goat anti-mouse IgG F(ab′)2 fragment (0.16 μg/ml) was added to plates and incubated 1.5 h at RT. ADA IgG (murine antibodies against the injected antibodies FluAB_wt and FluAB_MLNS) were then revealed with the appropriate substrate and read with a spectrophotometer. Data shown are the OD values (450 nm) obtained in each individual serum (n=5/group) collected 14 and 21 days after the antibody i.v. administration. Sera from naïve Tg32 mice (ctrl) were used as negative control.

[0280] Results are shown in FIG. 28. Surprisingly, the signal of murine serum IgG reacting against the FluAB-MLNS antibody was very low and corresponding to the signal detected in control, non-injected animals, while the ADA response measured in the serum of mice injected with FluAB_wt was, instead, significantly high, both 14 and 21 day after i.v. injection (FIG. 28A). In addition, the levels of ADA measured at day 14 post injection significantly and inversely correlated with the levels of circulating FluAB_wt (serum FluAB_wt levels decreased due to murine antibodies against FluAB_wt), while the levels of circulating FluAB-MLNS measured at the same time were indeed much higher and homogeneous (FIG. 28B).

[0281] In summary, these data indicate that surprisingly the anti-drug response (anti-drug antibodies; ADA), and, thus, the immunogenicity of FluAB_MLNS was decreased compared to FluAB_wt.

Example 10: Anti-Drug Antibody Response and Immunogenicity after s.c. Administration

[0282] To further confirm this surprising finding in more immunogenic settings, separate groups of TG32 mice (n=5) were injected with either FluAB-MLNS or FluAB_wt (5 mg/kg) subcutaneously (s.c.), which is generally considered a more immunogenic route of administration. Three weeks after s.c. administration, the levels of anti-drug antibodies were measured in the serum by mouse anti-drug specific ELISA (as described above in Example 9) in the serum of mice injected s.c. with either FluAB_wt or FLuAB_MLNS. As negative control, a pool of 10 sera from naïve, untreated animals was used.

[0283] Results are shown in FIG. 29. Despite the more immunogenic settings, animals treated s.c. with FluAB-MLNS still did not mount a humoral immunogenic response, as confirmed by the serum titer of anti-hIgG antibodies that was overlapping to the one detected in the serum of non-injected control animals. Conversely, the ADA titer in animals treated with FluAB_wt was clearly positive and measurable in all treated animals. An inverse correlation between the circulating levels of injected antibody and anti hIgG endogenous response was detected in the sera of mice injected with FluAB_wt only (not shown).

[0284] These data surprisingly show that antibody FluAB_MLNS exhibits less immunogenicity as compared to its parental antibody FluAB_wt.

TABLE-US-00005 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks FluAB_MLNS SEQ ID NO: 1 SYNAVWN CDRH1 SEQ ID NO: 2 RTYYRSGWYNDYAESVKS CDRH2 SEQ ID NO: 3 SGHITVFGVNVDAFDM CDRH3 SEQ ID NO: 4 RTSQSLSSYTH CDRL1 SEQ ID NO: 5 AASSRGS CDRL2 SEQ ID NO: 6 QQSRT CDRL3 SEQ ID NO: 7 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWN VH WIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPD TSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAF DMWGQGTMVTVSS SEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQK VL PGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 9 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNW Heavy chain IRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTS KNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFD MWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 10 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQK Light chain PGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC FluAB_wt SEQ ID NO: 11 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWN Heavy chain WIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPD TSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAF DMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FluAB_MLNS nucleic acid sequences SEQ ID NO: 12 CAAGTTCAGCTGCAGCAGAGCGGCCCCGGTCTGGTGA FluAB_MLNS VH nuc AGCCTAGCCAGACTCTGTCTTTAACTTGCGCCATCTCC GGCGACAGCGTGAGCAGCTACAACGCCGTCTGGAACT GGATTCGTCAGAGCCCTAGCAGAGGTTTAGAGTGGCT GGGTCGTACTTACTATCGTTCCGGCTGGTACAACGACT ACGCCGAGAGCGTGAAGTCTCGTATCACTATCAACCC CGATACTAGCAAGAACCAGTTCTCTTTACAGCTGAACA GCGTGACTCCCGAAGACACTGCCGTGTACTACTGCGC TCGTAGCGGCCACATCACTGTGTTCGGCGTGAATGTG GACGCCTTCGACATGTGGGGCCAAGGTACTATGGTCA CTGTGAGCAGC SEQ ID NO: 13 GACATCCAGATGACTCAGAGCCCTTCCTCTTTAAGCGC FluAB_MLNS VL nuc TAGCGTGGGCGATAGGGTCACTATCACTTGTCGTACT AGCCAGTCTTTAAGCTCCTACACTCACTGGTACCAGCA GAAGCCCGGTAAGGCCCCTAAGCTGCTGATCTACGCT GCCAGCAGCAGAGGCAGCGGAGTGCCTAGCAGATTT AGCGGCAGCGGTAGCGGCACTGACTTCACTCTGACAA TCAGCTCTTTACAGCCCGAAGACTTCGCCACTTACTAC TGCCAGCAGTCTCGTACTTTCGGCCAAGGTACTAAGGT GGAGATCAAG SEQ ID NO: 14 CAAGTTCAGCTGCAGCAGAGCGGCCCCGGTCTGGTGA FluAB_MLNS heavy AGCCTAGCCAGACTCTGTCTTTAACTTGCGCCATCTCC chain nuc GGCGACAGCGTGAGCAGCTACAACGCCGTCTGGAACT GGATTCGTCAGAGCCCTAGCAGAGGTTTAGAGTGGCT GGGTCGTACTTACTATCGTTCCGGCTGGTACAACGACT ACGCCGAGAGCGTGAAGTCTCGTATCACTATCAACCC CGATACTAGCAAGAACCAGTTCTCTTTACAGCTGAACA GCGTGACTCCCGAAGACACTGCCGTGTACTACTGCGC TCGTAGCGGCCACATCACTGTGTTCGGCGTGAATGTG GACGCCTTCGACATGTGGGGCCAAGGTACTATGGTCA CTGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCCGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGT GCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCA ACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GCGGGTTGAGCCCAAATCTTGTGACAAAACTCACACA TGCCCACCGTGCCCAGCACCTGAACTCCTGGCGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCACTCCCCGAAGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCC CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT CTCATGCTCCGTGCTGCATGAGGCTCTGCACAGCCACT ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 15 GACATCCAGATGACTCAGAGCCCTTCCTCTTTAAGCGC FluAB_MLNS light TAGCGTGGGCGATAGGGTCACTATCACTTGTCGTACT chain nuc AGCCAGTCTTTAAGCTCCTACACTCACTGGTACCAGCA GAAGCCCGGTAAGGCCCCTAAGCTGCTGATCTACGCT GCCAGCAGCAGAGGCAGCGGAGTGCCTAGCAGATTT AGCGGCAGCGGTAGCGGCACTGACTTCACTCTGACAA TCAGCTCTTTACAGCCCGAAGACTTCGCCACTTACTAC TGCCAGCAGTCTCGTACTTTCGGCCAAGGTACTAAGGT GGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTC ATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAC TGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAA GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT