Fusion proteins for the diagnosis, prophylaxis and treatment of infectious diseases

Abstract

The present invention inter alia relates to the use of phosphotidylserine or pathogenic sugar targeted therapeutics for the management and treatment of microbial infections, including Zika, Dengue, West Nile, Ebola, H1N1, enteroviruses, Leishmaniasis, Malaria and Coronaviruses SARS-COV. In an aspect, the invention concerns a fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin, and a peptide, protein, or antibody fragment binding to phosphatidylserine and/or a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe. Other aspects are described.

Claims

1. A fusion construct comprising an Ig-Fc domain or other protein scaffold, such as albumin or an antibody fragment binding to albumin, and a. a peptide, protein or antibody fragment binding to phosphatidylserine and/or b. a peptide or protein binding to and/or recognizing a PAMP expressed by a microbe.

2. A fusion construct, preferably according to claim 1, comprising an IgG-Fc domain or other protein scaffold and a. a recombinant human TIM1 fragment and/or b. a recombinant human CD209 fragment.

3. A fusion construct according to any of claims 1-2, comprising an IgG-Fc domain or other protein scaffold, and a. A recombinant Ig-like V-type domain of a human TIM1 and/or b. A recombinant C-type lectin domain of a human CD209 fragment.

4. A fusion construct according to claim 3, comprising an IgG-Fc domain or other protein scaffold, and a. Two or more recombinant Ig-like V-type domains from one or more human TIM1 and/or b. Two or more recombinant C-type lectin domains from one or more human CD209 fragment(s).

5. A fusion construct comprising a. A recombinant Ig-like V-type domain of a human TIM1 and b. A recombinant C-type lectin domain of a human CD209 fragment.

6. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and a. a recombinant human TIM1 fragment and/or b. a recombinant human CD209 fragment and wherein said fusion construct provides enhanced ADCC, ADCP and/or CDC.

7. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and a. a recombinant human TIM1 fragment and/or b. a recombinant human CD209 fragment and wherein said fusion construct additionally comprises the CDR regions according to SEQ ID No.: 54 - 59.

8. A fusion construct, preferably according to any of the preceding claims comprising an IgG-Fc domain or other protein scaffold and a. a recombinant human TIM1 fragment and/or b. a recombinant human CD209 fragment and wherein said fusion construct further comprises a Furin inhibitor.

9. The fusion construct according to any of the preceding claims, wherein said peptide, protein or antibody fragment is capable of binding to and/or stimulating an immune cell.

10. The fusion construct according to any of the preceding claims, wherein said TIM1 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 50-180 amino acid residues, 60-160 amino acid residues, 70-140 amino acid residues, 80-130 amino acid residues, 90-120 amino acid residues, 100-120 amino acid residues and 100-110 amino acid residues.

11. The fusion construct according to any of the preceding claims, wherein said CD209 fragment has a sequence length selected from the group consisting of 40-200 amino acid residues, 40-190 amino acid residues, 50-180 amino acid residues, 60-170 amino acid residues, 70-160 amino acid residues, 80-150 amino acid residues, 90-150 amino acid residues, 100-150 amino acid residues, 110-150 amino acid residues, 120-150 amino acid residues and 130-140 amino acid residues.

12. The fusion construct according to any of the preceding claims, wherein said TIM1 and/or CD209 fragment has a sequence homology of at least 70%, alternatively 75%, alternatively 80%, alternatively 85%, alternatively 90%, alternatively 95% to wildtype TIM1 or CD209.

13. The fusion construct according to any of the preceding claims, wherein said TIM1 and/or CD209 fragment has intact TIM1 and/or CD209 function.

14. The fusion construct according to any of the preceding claims, wherein said IgG-Fc domain is an IgG3-Fc domain.

15. The fusion construct according to any of the preceding claims, comprising additionally at least one of the following: a) An IgG3, wherein the hinge sequence has been replaced, preferably with an IgG4 or IgG1 hinge sequence; b) CDR regions according to SEQ ID No.: 54 - 59; and/or c) A furin inhibitor.

16. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1 and/or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

17. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

18. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or preferably at least 8 disulfide bonds.

19. The fusion construct according to any of the preceding claims, wherein said fusion construct is capable of binding to a target, and wherein said target is a mannan, a high-mannose containing structure, a fucan, a phospholipid phosphatidylserine and/or CD3.

20. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A protein fragment comprising or consisting of a sequence according to SEQ ID No.: 1, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to a sequence according to SEQ ID No.: 1, and b. a protein fragment comprising or consisting of a sequence according to SEQ ID No.: 3 or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to a sequence according to SEQ ID No.: 3.

21. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and ii. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43,, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and b. A second chain comprising iii. a sequence according to SEQ ID No.: 1or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

22. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 9 or a sequence according to SEQ ID No.: 43, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

23. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 11 or a sequence according to SEQ ID No.: 45, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 13 or a sequence according to SEQ ID No.: 47, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

24. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and v. a linker sequence, preferably according to SEQ ID No.: 41, and vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

25. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 3 and/or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences and v. a linker sequence preferably according to SEQ ID No.: 41, and vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

26. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and v. a linker sequence preferably according to SEQ ID No.: 41, and vi. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

27. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 16 or 17, or SEQ ID No.: 67, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iii. a linker sequence preferably according to SEQ ID No.: 41, and iv. a sequence according to any of the sequences selected among SEQ ID No.: 18 - 35, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising v. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, vi. a linker sequence preferably according to SEQ ID No.: 41, and vii. a sequence according SEQ ID No.: 14 or 15, or SEQ ID No.: 66, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

28. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a linker.

29. The fusion construct according to any of the preceding claims, wherein said linker is selected among a (GGGGS)3 linker (SEQ ID NO. 41), a (GGGGS)4 linker (SEQ ID NO. 70), a (GGGGS)5 linker (SEQ ID NO. 71) and a (GGGGS)6 linker (SEQ ID NO. 72).

30. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises at least one free cysteine residue, at least two free cysteine residues, at least three free cysteine residues, at least four free cysteine residues, at least five free cysteine residues or preferably at least six free cysteine residues.

31. The fusion construct according to any of the preceding claims, wherein said free cysteine allows interaction with a drug and/or a payload.

32. The fusion construct according to any of the preceding claims, wherein said payload is a furin inhibitor.

33. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a A339C mutation, a S337C mutation and/or a K340C mutation.

34. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a sequence selected among any of the sequences SEQ ID No.: 36, 37, SEQ ID No.: 38, 39, 40, 42, 44 or 46.

35. The fusion construct according to any of the preceding claims, wherein said fusion construct is an IgG1, IgG2, IgG3 or an IgG4.

36. The fusion construct according to any of the preceding claims, wherein said fusion construct is an IgG, IgM, IgA, IgD or an IgE.

37. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a null fc.

38. The fusion construct according to any of the preceding claims, wherein said null fc comprises an Ala substitution at position 234 and/or Ala substitution at 235, and/or N297A, and/or a K322A mutation.

39. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a heterodimerization domain.

40. The fusion construct according to any of the preceding claims, wherein said heterodimerization domain comprises a sequence according to SEQ ID No.: 48, 49 or 50.

41. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a heterodimerization mutation.

42. The fusion construct according to any of the preceding claims, wherein said heterodimerization mutation is an F405L, R409K and/or K409R mutation.

43. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to this sequence.

44. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

45. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a sequence according to SEQ ID No.: 38, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and b. A second chain comprising iii. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and iv. a sequence according to SEQ ID No.: 40, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

46. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a linker sequence according to SEQ ID No.: 41, and iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences b. A second chain comprising iv. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and v. a linker sequence according to SEQ ID No.: 41, and vi. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

47. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 3 or SEQ ID No.:4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a linker sequence according to SEQ ID No.: 41, and iii. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences b. A second chain comprising iv. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and v. a linker sequence according to SEQ ID No.: 41, and vi. a sequence according to SEQ ID No.: 65, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences.

48. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises: a. A first chain comprising i. a sequence according to SEQ ID No.: 1 or SEQ ID No.: 2, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and ii. a linker sequence according to SEQ ID No.: 41, and iii. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8 b. A second chain comprising iv. a sequence according to SEQ ID No.: 3 or SEQ ID No.: 4, or a sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to one of these sequences, and v. a linker sequence according to SEQ ID No.: 41, and vi. a sequence according to SEQ ID No.: 65, wherein said sequence ID No.: 65 comprises one or more of the mutations of table 8.

49. The fusion construct according to any of the preceding claims, wherein the ratio of fusion construct to said drug and/or payload is selected among 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

50. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises a kappa light chain according to SEQ ID No.: 51 or a lambda light chain according to SEQ ID No.: 52 or 53.

51. A fusion construct, preferably according to any of the preceding claims, wherein said fusion construct is an IgG3 construct, and wherein said IgG3 construct comprises a hinge region, wherein said hinge region has been modified.

52. The fusion construct according to claim 51, wherein said hinge region comprises a sequence having a total of at least 10% identity, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the sequence according to SEQ ID No.: 6 or SEQ ID No.: 8.

53. The fusion construct according to any of the preceding claims, wherein said fusion construct comprises the sequence according to SEQ ID No.: 5, 7, 9, 10, 11, 12 and/or 13.

54. The fusion construct according to any of the preceding claims, wherein said hinge region comprises at least one free cysteine residue, at least two free cysteine residues or preferably at least three free cysteine residues.

55. The fusion construct according to any of the preceding claims, wherein said hinge region comprises a S228P mutation.

56. The fusion construct according to any of the preceding claims, wherein said hinge region comprises a sequence according to SEQ ID No.: 6 and/or SEQ ID No.: 8 and/or SEQ ID No.: 68.

57. The fusion construct according to any of the preceding claims, wherein said fusion construct is used to detect phosphatidylserine.

58. The fusion construct according to any of the preceding claims, wherein said fusion construct is used to detect phosphatidylserine in the blood of a subject.

59. The fusion construct according to claim 57, wherein said fusion construct comprises a sequence according to SEQ ID No.: 1, and/or a sequence according to SEQ ID No.: 2.

60. The fusion construct according to any of the preceding claims, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties.

61. The fusion construct according to any of the preceding claims, wherein said fusion construct is used to detect C-type lectin binding mannan or fucan moieties in the blood of a subject.

62. The fusion construct according to claim 60, wherein said fusion construct comprises a sequence according to SEQ ID No.: 3 and/or a sequence according to SEQ ID No.: 4.

63. A fusion construct, a fusion protein or an antibody comprising the constant region(s) of IgG3 and a hinge, wherein said hinge preferably is selected among an IgG1 or IgG4 hinge.

64. The fusion construct, fusion protein or antibody according to any claim 63, comprising one or more heterodimerization mutations.

65. The fusion construct, fusion protein or antibody according to claim 64, comprising heterodimerization mutations involving or including positions 405 and/or 409 (EU numbering).

66. IgG3 homodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.

67. IgG3 heterodimer comprising a hinge region, wherein said hinge region comprises a sequence selected among SEQ ID No.: 6, 8 and 68.

68. IgG3 according to any of claims 66-67, wherein said IgG3 comprises a mutation at position 405 and/or position 409.

69. IgM heterodimers obtainable by changing the charge pairs of the CH2 and/or CH4 domains.

70. IgM heterodimers according to claim 69, comprising one or more of the mutations of Table 8.

71. The IgM according to any claims 69-70, wherein said IgM comprises a sequence according to SEQ ID No.: 64 and/or 65.

72. A fusion construct according to any of claims 1-65, wherein said fusion construct comprises an IgG3 homodimer, an IgG3 heterodimer and/or an IgM heterodimer according to any of claims 66-71.

73. The fusion construct according to any of the preceding claims, wherein said fusion construct is for use in the treatment of an infection.

74. The fusion construct according to claim 73, wherein said infection is an infection caused by a virus, such as Coronaviruses SARS-COV, a parasite, a bacteria, a fungi or a protozoan.

75. The fusion construct according to claims 74, wherein said virus is selected among an arborvirus, Zika virus, Dengue virus, West Nile virus, Ebola virus, influenza virus, influenza virus H1N1, Chikungunya virus, enterovirus, Coronavirus SARS-COV-2 and Coronaviruses SARS-COV.

76. The fusion construct according to claim 74, wherein said bacteria is selected among mycobacterium tuberculosis and mycobacterium leprae.

77. The fusion construct according to claim 74, wherein said parasite is selected among Leishmaniasis and Malaria.

78. Use of a fusion construct according to any of claims 1-71, for the treatment of an infection.

79. Use according to claim 78, wherein said infections are selected among viral, bacterial and protozoan infections.

80. Use according to any claims 78-79, wherein the treatment comprising administration of the fusion construct with an administration form selected among subcutaneous, intradermal, intramuscular, oral and nasal.

81. Use of IgG4 or a part of IgG4 for payload delivery, wherein said IgG4 has been modified to comprise no Fc or wherein the activity of the Fc of said IgG4 has been nullified or diminished by one or more mutations.

82. The use according to any of claims 78-81, wherein said IgG4 comprises one or more heterodimerization mutations.

83. The use according to any of claims 81-82, wherein said IgG4 comprises one or more Cys mutations, preferably thereby allowing site specific conjugation.

84. The use according to any of claims 81-83, wherein said IgG4 comprises a Cys at position 339 (EU numbering).

85. A vaccine comprising a fusion construct according to any of claims 1-65.

86. A vaccine comprising a mannan, a high mannose containing structure, a fucan and/or a phospholipid phosphatidylserine (PS).

87. The vaccine according to claim 85 or 86, further comprising β-glucan.

88. The vaccine according to any of claims 85-87, for the prevention and/or treatment of an infection.

89. The vaccine according to claim 88, wherein said infection is caused by a virus, preferably according to claim 75, and/or Coronaviruses SARS-COV, a parasite, preferably according to claim 77, a bacteria, preferably according to claim 76, a fungi or a protozoan.

90. The fusion construct according to any of claims 1-65 and/or vaccine according to any of claims 85-89, wherein said fusion construct and/or vaccine allows administration through a route selected among subcutaneous administration, intradermal administration, intramuscular administration, oral administration and/or nasal administration.

91. A composition comprising a fusion construct according to any of claims 1-65, optionally comprising one or more excipients such as diluents, binders or carriers.

92. A method of treating and/or preventing an infection in a subject, comprising a step of administration of a fusion construct according to any of claims 1-65 and/or a vaccine according to any of claims 85-89 and/or a composition according to any of claims 90-91.

93. A method of screening and/or monitoring progression of a disease in a subject, wherein said method comprises the following steps: i. Providing a blood sample from said subject. ii. Contacting said blood sample with a fusion construct according to any of claims 1-65.

94. An isolated nucleic acid molecule encoding a fusion construct according to any of claims 1-65.

95. A recombinant vector comprising the nucleic acid molecule of claim 94.

96. A host cell comprising the recombinant vector of claim 95.

97. A method for the production of a fusion construct according to any of the precedent claims comprising a step of culturing the host cell according to claim 96 in a culture medium under conditions allowing the expression of the fusion construct and separating the fusion construct from the culture medium.

Description

FIGURES

[0317] FIG. 1 shows TIM1 and CTLD constructs with enhanced ADCC, ADCP and CDC.

[0318] FIG. 2 shows TIM1 and CTLD constructs with T cell engager activity.

[0319] FIG. 3 shows TIM1 and CTLD constructs with furin inhibitor payload.

[0320] FIG. 4 shows TIM1 and CTLD constructs with IgM effector function.

[0321] FIGS. 5a and 5b shows SEC-HPLC analysis of VP019 and VP020 respectively.

[0322] FIG. 6a shows that VP025 consists of 4 distinct peaks. FIG. 6b: intact mass spectrometry Fraction 3 from peak 3 (VP025-F3). FIG. 6c: intact mass spectrometry: Fraction 4 from peak 4 (VP025-F4).

[0323] FIGS. 7a and 7b show the schematics and purity of VP300 and VP301 by SEC-HPLC.

[0324] FIG. 8 shows binding of SARS-CoV-2 S protein (D614G)

[0325] FIG. 9 shows binding of VP025-CT to viral proteins

[0326] FIGS. 10a and 10b show binding curves of VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens.

[0327] FIG. 11 shows the structure of Hexa-D-arginine linker-compound and Decanoyl-RVKR-CMK linker compound

[0328] FIG. 12 shows the reaction scheme for the Decanoyl-RVKR-CMK linker compound

[0329] FIGS. 13a and b shows a mass-spectrogram demonstrating that conjugation of furin inhibitor payload to VP020 has been accomplished.

[0330] FIG. 14a shows a neutralization assay for selected fusion proteins of the invention. For more details see example 16.

[0331] FIG. 14b shows a neutralization assay for selected fusion protein of the invention. For more details see example 17.

[0332] All cited references are incorporated by reference.

[0333] The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.

[0334] Unless otherwise mentioned, all percentages are in weight/weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 8.0.

EXAMPLES

Example 1: Selection of Recombinant Human TIM1 Fragment

[0335] Construct V-TIM1-1 was selected as residues 21-125 of the full length TIM-1 sequence (https://www.uniprot.org/uniprot/Q96D42), and V-TIM1-2 was selected as residues 21-127. V-TIM1-2 contains an extra two Pro residues at the C-terminal domain boundary.

TABLE-US-00003 Sequences of Recombinant human TIM1 fragment Construct Sequence Mw Predicted pl V-TIM1-1 SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTC QNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENT AVSDSGVYCCRVEHRGWFNDMKITVSLEIV SEQ ID No.: 1 11.6 KDa 8.26 V-TIM1-2 SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTC QNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENT AVSDSGVYCCRVEHRGWFNDMKITVSLEIVPP SEQ ID No.: 2 11.8 KDa 8.26

Example 2: Selection of Recombinant Human C-Type Lectin Domain (CTLD) Fragment of DC-SIGN (CD209)

[0336] Construct V-CTLD-1 was selected as residues 250-385 of the full length DC-SIGN sequence (https://www.uniprot.org/uniprot/Q9NNX6), and V-CTLD-2 was selected as residues 254-383. V-CTLD-1 contains 4 internal disulfide bonds, whereas V-CTLD-2 contains 3 internal disulfide bonds.

TABLE-US-00004 Sequences of Recombinant human CTLD fragment of DC-SIGN Construct Sequence MW Predicted pl V-CTLD-1 ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACK EVGAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLN QEGTWQWVDGSPLLPSFKQYWNRGEPNNVGEEDCA EFSGNGWNDDKCNLAKFWICKKSAASCS SEQ ID No.: 3 15.7 KDa 5.12 V-CTLD-2 HPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVG AQLVVI KSAEEQN FLQLQSSRSN RFTWMG LSDLNQEG TWQWVDGSPLLPSFKQYWN RGEPN NVG EEDCAEFSG NGWNDDKCNLAKFWICKKSAAS SEQ ID No.: 4 15.0 KDa 5.08

Example 3: Design of TIM-1 and CTLD Constructs With IgG3 Effector Functions

[0337] Among all human IgG subclasses, IgG3 has the highest effector functions in terms of ADCC, ADCP and CDC (https://www.frontiersin.org/articles/10.3389/fimmu.2014.00520/full). IgG3 has not typically been used for therapeutics because of the short serum half-life due to proteolytic cleavage of the prolonged hinge region between the CH1 and CH2 domains. To utilize the strong effector functions of the IgG3 subclass, the V-IGG3 construct was designed where the IgG3 hinge (LKTPLGDTTHTPEPKSCDTPPPCPRCPAP) (SEQ ID NO. 6) was replaced with an IgG4 hinge sequence containing an IgG4 hinge S228P mutation to prevent Fab arm exchange (SKYGPPCPPCPAP) (SEQ ID NO. 8) or an IgG1-like hinge (KTGDTTHTCPRCPAP) (SEQ ID NO. 68).

[0338] Heterodimeric V-IGG3 constructs were designed based on including K409R (on one half-antibody) and F405L (on second antibody) mutation in the CH3 domains (https://www.nature.com/articles/nprot.2014.169). Each half antibody is first generated as a single homodimer, then mixed together and allowed to recombine as heterodimers under reducing and oxidizing conditions. The resulting sequences are noted as V-IGG3-A and V-IGG3-B and pair together, or V-IGG3-D and V-IGG3-E that pair together. Sequences are found in Table 3, including truncated version that include a (GGGGS)3 linker (SEQ ID NO. 41) to replace the CH1 domains.

TABLE-US-00005 Modified IgG3 domains Construct Sequence Notes WT huIgG3 CH1-CH2-CH3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVN H KPSNTKVDKRVE LKTPLG DTTHTPEPKSCDTPPPCPR CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVM HEALHNRFTQKSLSLSPGK SEQ ID No.: 5 Hinge region underlined as SEQ ID No.: 6 V-IGG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 7 Hinge region underlined (SEQ ID No.: 8) with IgG4 hinge S228P mutation in bold V-lGG3-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 9 Same as above, but with linker and fc only V-IGG3-A ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 10 K409R V-lGG3-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL Same as above, but with linker and fc only YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K SEQ ID No.: 11 V-IGG3-B ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 12 F405L V-IGG3-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKL TVDKSRWQQGNI FSCSVM HEALH NRFTQKSLSLSPG K SEQ ID No.: 13 Same as above, but with linker and fc only V-IGG3-C ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 42 Utilizes an IgG1-like hinge V-IGG3-C-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 43 Same as above, but with linker and fc only V-IGG3-D ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVH NAKTKPREEQYNSTFRVVSVL TVLHQDWLNG KEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPM LDSD K409R GSFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 44 V-IGG3-D-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 45 Same as above, but with linker and fc only V-IGG3-E ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVH NAKTKPREEQYNSTFRVVSVL TVLHQDWLNG KEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK SEQ ID No.: 46 F405L V-IGG3-E-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK SEQ ID No.: 47 Same as above, but with linker and fc only

[0339] TIM1 and CTLD fusion proteins were designed with the modified IgG3-Fc domains and are depicted in FIG. 1 and Table 4.

TABLE-US-00006 Sequences of TIM-1 and CTLD constructs with enhanced ADCC, ADCP and CDC Construct Chain 1 Chain 2 VIT-300 (V-TIM1-1/V-TIM1-2) -V-IGG3-Fc/V-IGG3-C-Fc Same as Chain 1 VIT-301 (V-CTLD-1/V-CTLD-2) -V-IGG3-Fc/ V-IGG3-C-Fc Same as Chain 1 VIT-302 (V-TIM1-1/V-TIM1-2) -V-IGG3-A-Fc/ V-IGG3-D-Fc (V-CTLD-1/V-CTLD-2) -V-IGG3-B-Fc/ V-IGG3-E-Fc

Example 4. Design of TIM-1 and CTLD Constructs With T Cell Engaging Activity

[0340] Additional constructs were designed to engage T cell effector functions by fusing the TIM-1 and CTLD with a single anti-CD3 scFv. The designs are shown in FIG. 2 and Table 5.

TABLE-US-00007 Sequences of TIM-1 and CTLD constructs with T cell engager activity Construct Chain 1 Chain 2 VIT-303 (V-TIM1-1/V-TIM1-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc/V-IGG2-D-Fc) (V-TIM1-1/V-TIM1-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-304 (V-CTLD-1/V-CTLD-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) (V-CTLD-1/V-CTLD-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-305 (V-TIM1-1/V-TIM1-2)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc) (V-CTLD-1/V-CTLD-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/ V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) VIT-306 (V-TIM1-1/V-TIM1-2)-(V-IGG4-B-Fc/ V-IGG2-B-Fc/ V-IGG2-E-Fc)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(anti-CD3 scFv) (V-CTLD-1/V-CTLD-2)-GGGGSGGGGSGGGGS (SEQ ID No.: 41)-(V-IGG4-A-Fc/ V-IGG2-A-Fc /V-IGG2-D-Fc)

TABLE-US-00008 Anti-CD3 scFv sequences are described below Construct Sequence VCD3-H1L1 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 18) VCD3-H1L1-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 19) VCD3-H1L2 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 20) VCD3-H1L2-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 21) VCD3-H1L3 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLOQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 22) VCD3-H1L3-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 23) VCD3-H2L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYD DHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 24) VCD3-H2L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 25) VCD3-H2L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQG RVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 26) VCD3-H2L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 27) VCD3-H2L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 28) VCD3-H2L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 29) VCD3-H3L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 30) VCD3-H3L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 31) VCD3-H3L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 32) VCD3-H3L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 33) VCD3-H3L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO: 34) VCD3-H3L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO: 35)

Example 5. Design of TIM-1 and CTLD Constructs With Furin Inhibitor Payload Delivery

[0341] Site specific addition of drug payloads to the antibody Fc region was devised by analysis of the co-crystal structure of a human IgG1 Fc with the 3-helix bundle of bacterial protein A (PDB structure 5U4Y https://www.rcsb.org/sequence/5U4Y). Computational modelling revealed that A339C would have a stabilizing effect to the structure and S337C or K340C would have a neutral effect to the stability of the Fc domain. A339C was chosen as the site for site specific conjugation.

TABLE-US-00009 IgG4 sequences with engineered free cys for site specific payload conjugation Construct Sequence Notes V-IGG4-ADC-A ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK SEQ ID No.: 37 S228P FALA (F234A, L235A) K322A Naturally contains F405 and R409 A339C V-IGG4-ADC-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF Same as above, but with linker and fc only V-IGG4-ADC-B ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK SEQ ID No.: 39 S228P FALA (F234A, L235A) K322A F405L, R409K A339C V-IGG4-ADC-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL LYSKLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK SEQ ID No.: 40 Same as above, but with linker and fc only

[0342] TIM1 and CTLD fusion proteins with Fc domain with payload conjugation sites were designed and are shown in FIG. 3 and Table 8.

TABLE-US-00010 Sequences of TIM1 and CTLD therapeutic proteins with free cys for payload conjugation Construct Chain 1 Chain 2 VIT-307 (V-TIM1-1/V-TIM1-2) -V-IGG4-ADC-A-Fc Same as Chain 1 VIT-308 (V-CTLD-1/V-CTLD-2) -V-IGG4-ADC-A-Fc Same as Chain 1 VIT-309 (V-TIM1-1/V-TIM1-2) -V-IGG4-ADC-A-Fc (V-CTLD-1/V-CTLD-2) -V-IGG4-ADC-B-Fc

Example 6. Furin Linkers

[0343] Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) (SEQ ID NO. 81) or hexa-D-arginine (D6R) were linked to TIM-1 and CTLD constructs using cleavable linkers such as acid sensitive N-acyl-hydrazone or enzyme sensitive malemeide-conjugated dipeptides, valine-alanine, valine-citrulline, or phenylalanine-Lysine.

[0344] Acid sensitive linkers are cleaved in the lysosome acidic environment after internalization of the construct. This strategy has been used in two approved ADCs, Gemtuzumab ozogamicin and Inotuzumab ozogamicin. Lysosomal protease sensitive dipeptides release the drug after cleavage by proteases such as cathepsin B- lysosomal protease. This type of linker chemistry has been used for FDA approved Brentuximab vedotin.

[0345] Linkage to the polypeptide of antibodies is done through the nucleophilic groups of lysine or cysteine by random conjugation, generating a heterogeneous mixture of conjugates, or by site-directed conjugation to engineered cysteines, reducing the heterogeneity of the product to an antibody-drug ratio (ADR) of 1 or 2.

[0346] The nucleophilic reactivity of the thiol functionality of a Cys residue to a maleimide group is about 1000 times higher compared to any other amino acid functionality in a protein, such as amino group of lysine residues or the N-terminal amino group. Thiol specific functionality in maleimide reagents may react with amine groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London).

[0347] The first FDA approved site-directed ADC through engineered cysteines was vadastuximab talirine (Seattle Genetics).

TABLE-US-00011 Linker Chemistry Residue Spacer 1 Linker Spacer 2 Drug Cysteine, engineered Cysteine Maleimidocaproyl (mc), maleimidomethyl VC, VA, PL para-amino benzyloxycarbonyl (PABC) hexa-D-arginine, dec-RVKR-cmk cyclohexane- 1-carboxylate (comprising SEQ ID NO. 81) Lysine NA N-acyl-hydrozone, N-succinimidyl-4-(2-pyridyldithio) Butanoate-disulfide (SPDB-disulfide), maleimidomethyl cyclohexane-1-carboxylate, sulfo-SPDB NA hexa-D-arginine, dec-RVKR-cmk (comprising SEQ ID NO. 81) Spacer 1: The purpose of the mc spacer is to provide enough room so that the vc group can be recognized by cathepsin B, which cleaves the citrulline-PABC amide bond. Spacer 2: Self-immolative spacer

Example 7. Fusion Proteins With IgM Constant Regions

[0348] IgM molecules have robust Fc effector functions, particularly with CDC. IgM molecules naturally homodimerize and then covalently associate into pentamers or hexamers. IgM do not contain hinge regions like IgG molecules and instead contain an extra CH domain (CH1-CH2-CH3-CH4). The homodimeric heavy chains come together at the CH2 and CH4 domains. Based on visual analyses of the crystal structure of the murine IgM CH2 domain (pdb 4JVU), the crystal structure of the murine IgM CH4 domain (pdb 4JVW), and a sequence alignment of the human IgM CH2 and CH4 sequences with the homologous mouse sequences, mutations were designed to induce IgM heavy chain heterodimerization by inducing charge differences at the homodimerization interfaces.

[0349] Sequence of human IgM constant region, numbered residues 1-453 by uniprot (www_uniprot.org/uniprot/P01871):

TABLE-US-00012 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDI SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKN VPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLR EGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLT TYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATIT CLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTV SEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAG TCY SEQ ID No.: 64

[0350] Sequence of IgM CH2-CH3-CH4 which can used for fusing to antibody fragments (Fab, scFv, VHH, etc) or targeting proteins (TIM-1, CTLD/DC-SIGN) for adding IgM effector functions (residues 105-453): V-IGM

TABLE-US-00013 VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGL TFQQONASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLYVTDLTTY DSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFT CTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCL VTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSE EEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTC Y SEQID No.: 65

[0351] Based on the structural analysis, the underlined residues K131 and Q135 were found to be in close proximity in the CH2:CH2 interface, and residues T354 and E385 were found to be in close proximity in the CH4:CH4 interface. The following mutations were made to alter the charge pattern in V-IGM-A and V-IGM-B to induce heterodimer formation of A:B and repel the formations of A:A or B:B.

TABLE-US-00014 IgM constant region mutations to induce heavy chain heterodimer formation Position Wildtype residue in V-IGM V-IGM-A V-IGM-B 131 K K/R/H D/E 135 Q K/R/H D/E 354 T D/E K/R/H 385 E D/E K/R/H

[0352] TIM1 and CTLD fusion proteins with IgM effector functions were designed and shown in FIG. 4 and Table 11.

TABLE-US-00015 Sequences of TIM1 and CTLD therapeutic proteins with IgM effector functions Construct Chain 1 Chain 2 VIT-310 (V-TIM1-1/V-TIM1-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41)-V-IGM Same as Chain 1 VIT-311 (V-CTLD-1/V-CTLD-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41) -V-IGM Same as Chain 1 VIT-312 (V-TIM1-1/V-TIM1-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41) -V-IGM-A (V-CTLD-1/V-CTLD-2) -GGGGSGGGGSGGGGS (SEQ ID No.: 41)-V-IGM-B

Example 8: Expression and Purification of VP011-VP020

[0353] The 10 proteins from [Table 1 and FIGS. 1-3] were expressed and batch purified form 2.5 mL ExpiCHO cultures using Protein A/G magnetic agarose beads. Expression yields and % monomeric purity are shown in Table 12.

TABLE-US-00016 Small scale expression of protein constructs Protein Name Gene Name Description Fc domain Format Yield (.Math.g) %Purity by SEC-HPLC VP011 TM-G1 V-TIM1-1 with V-IGG1-Fc IgG1 VIT-300-IgG1 143 28.6 VP012 TM-G3 V-TIM1-1 with V-IGG3-D-Fc IgG3 VIT-300-IgG3 150 34 VP013 CT-G1 V-CTLD-1 with IgG1-Fc IgG1 VIT-301-IgG1 209 56.5 VP014 CT-G3 V-CTLD-1 with V-IGG3-E-Fc IgG3 VIT-301-IgG3 225 73.4 VP015 TM-G4-A V-TIM1-1 with V-IgG4-A-Fc IgG4 VIT-300-IgG4 113 11 VP016 TM-G4-A-F V-TIM1-1 with V-IgG4-A-Fc-CD3 (H3L3) IgG4 73 16.8 VP017 CT-G4-B V-CTLD-1 with V-IgG4-B-Fc IgG4 VIT-301-IgG4 386 73.1 VP018 CT-G4-B-F V-CTLD-1 with V-IgG4-B-Fc-CD3 (H3L3) IgG4 290 74.4 VP019 TM-G4-A-DC V-TIM1-1 with V-IGG4-ADC-A-Fc IgG4 VIT-307 77 11.5 VP020 CT-G4-B-DC V-CTLD-1 with V-IGG4-ADC-B-Fc IgG4 VIT-308 645 60.9

[0354] Larger scale preps were done in ExpiCHO cells for VP011 (100 mL), VP012 (100 mL), VP013 (100 mL), VP014 (100 mL), VP019 (1L) and VP020 (250 mL). VP011, VP019 and VP020 were purified by MabSelect SuRe protein A resin column chromatography. VP012, VP013 and VP014 were purified by HiTrap Protein G resin column chromatography. Expression yields and % monomeric purity are shown in Table 13.

TABLE-US-00017 Larger scale expression of protein constructs Protein Name Gene Name Description Fc domain Format Yield (mg) %Purity by SEC-HPLC VP011 TM-G1 V-TIM1-1 with V-IGG1-Fc IgG1 VIT-300-IgG1 0.78 78.5.sup.∗ VP012 TM-G3 V-TIM1-1 with V-IGG3-D-Fc IgG3 VIT-300-IgG3 9.18 68.0.sup.∗ VP013 CT-G1 V-CTLD-1 with IgG1-Fc IgG1 VIT-301-IgG1 15.6 59.3 VP014 CT-G3 V-CTLD-1 with V-IGG3-E-Fc IgG3 VIT-301-IgG3 17.03 49.0.sup.∗ VP019 TM-G4-A-DC V-TIM1-1 with V-IGG4-ADC-A-Fc IgG4 VIT-307 12.73 60.7.sup.∗ VP020 CT-G4-B-DC V-CTLD-1 with V-IGG4-ADC-B-Fc IgG4 VIT-308 19.18 91.9 .sup.∗multimer peak

[0355] The sequences of the expressed recombinant proteins are shown in table 14.

TABLE-US-00018 Sequences of expressed recombinant proteins. Protein Name Description Sequence VP011 V-TIM1-1 with V-IGG1-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 82) VP012 V-TIM1-1 with V-IGG3-D-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSRLTVDKSRW QQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 83) VP013 V-CTLD-1 with IgG1-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQG NIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 84) VP014 V-CTLD-1 with V-IGG3-E-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLLYSKLTVDKSRW QQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 85) VP015 V-TIM1-1 with V-IgG4-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 86) VP016 V-TIM1-1 with V-IgG4-A-Fc-CD3 (H3L3) SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQSPGQCLEWMGYINPSRGYTNYNQKFQGRVTMTTDKSTSTAYMELSSLRSE DTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSElQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPR RWIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGC GTKLEIK (SEQ ID NO: 87) VP017 V-CTLD-1 with V-IgG4-B-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 88) VP018 V-CTLD-1 with V-IgG4-B-Fc-CD3 (H3L3) ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHW VRQSPGQCLEWMGYINPSRGYTNYNQKFQGRVTMTTDKSTSTAYMELSSLRSE DTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSEIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPR RWIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGC GTKLEIK (SEQ ID NO: 89) VP019 V-TIM1-1 with V-IGG4-ADC-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 90) VP020 V-CTLD-1 with V-IGG4-ADC-B-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 91) VP300 V-TIM1-1 and V-CTLD-1 with V-IGG4-ADC-A-Fc SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYR KDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVGG GGSGGGGSGGGGSERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEV GAQLVVIKSAEEQNFLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSF KQYWNRGEPNNVGEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCS GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 92) VP301 V-CTLD-1 and V-TIM1-1 with V-IGG4-ADC-A-Fc ERLCHPCPWEWTFFQGNCYFMSNSQRNWHDSITACKEVGAQLVVIKSAEEQN FLQLQSSRSNRFTWMGLSDLNQEGTWQWVDGSPLLPSFKQYWNRGEPNNV GEEDCAEFSGNGWNDDKCNLAKFWICKKSAASCSGGGGSGGGGSGGGGSSV KVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKD TRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIV GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCAVSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 93)

Example 9: Preparation of VP019 Fractions and VP025 Heterodimer

[0356] SEC-HPLC analysis of VP019 (FIG. 5a) shows that VP019 form the expected homodimer (Fraction F6) and multimer .sup.~600 kDa (Fraction F2). The fractions were purified out and assessed separately for functional activity (VP019-F6 and VP019-F2, respectively named).

[0357] FIG. 5b: SEC-HPLC analysis of VP020.

[0358] The protein product VP025, which is a heterodimer of VP019 and VP020, was generated by co-expressing Genes TM-G4-A-DC and CT-G4-B-DC in ExpiCHO cells and purified by MabSelect SuRe protein A resin column chromatography. The resulting co-transfected sample product was named VP025-CT. FIG. 6a shows that VP025-CT contains 4 distinct peaks which were purified out and assessed separately by intact mass spectrometry. The fractions from Peak 1 and Peak 2 showed a number of species. Fraction 3 from peak 3 (VP025-F3) showed a mass of 86,721 Da, corresponding to a VP020 homodimer (FIG. 6b). Fraction 4 from peak 4 (VP025-F4) showed a mass of 83,881 Da, corresponding to the properly formed VP019/VP020 heterodimer, at an approximate purity of 78% (FIG. 6c).

Example 10: Expression and Purification of VP300 and VP301

[0359] Two additional bispecific molecules containing the TIM-1 and CTLD domains on a single polypeptide chain (Table 15) were generated in 100 mL ExpiCHO cells and purified by MabSelect SuRe protein A resin column chromatography. The schematics and purity by SEC-HPLC are shown in FIGS. 7a and 7b.

[0360] The sequences of the expressed recombinant proteins are shown in table 14.

TABLE-US-00019 Larger scale expression of protein constructs Protein Name Gene Name Description Fc domain Yield (mg) VP300 TM-CT-G4-A-DC V-TIM1-1 and V-CTLD-1 with V-IGG4-ADC-A-Fc IgG4 2 VP301 CT-TM-G4-A-DC V-CTLD-1 and V-TIM1-1 with V-IGG4-ADC-A-Fc IgG4 2

Example 11: Binding of VP019, VP020 and VP025 to SARS-COV2 S Protein

[0361] The binding of VP019-F2, VP020 and VP025-CT to SARS-Cov-2 S D614G was investigated by ELISA. This protein is representative of the dominant SARS-COV-2 strain in early 2020. All ELISA assays in this and subsequent examples were done in the presence of 2.5 mM CaCl.sub.2 since DC-SIGN is known to use calcium at the binding site. Binding curves are shown in FIG. 8 and EC50 values are shown in Table 16. VP019-F2 had unexpectedly high binding to SARS-COV2 S Protein, while VP020 had modest binding.

TABLE-US-00020 EC50 for Binding of VP019, VP020 and VP025 to SARS-COV2 S Protein EC50 (mg/mL) EC50 (nM) VP019-F2 0.0009 10.6 VP020 0.0556 641.0 VP025-CT 0.0069 82.5

Example 12: Binding of VP025-CT to Wide Range of Viral Antigens

[0362] The binding of VP025-CT to many diverse viral surface protein antigens was investigated by ELISA. Binding curves are shown in FIG. 9 and EC50 values are shown in Table 17.

TABLE-US-00021 EC50 for binding of VP025-CT to several viral antigens EC50 (mg/mL) EC50 (nM) West Nile Virus Envelope Protein 0.0049 57.9 Chikungunya Virus E1 0.0102 121.5 Human RSV (A) Glycoprotein G 0.0696 829.6 Influenza A H1N1 HA 0.0062 73.9 SARS-Cov-2 S D614G 0.0040 47.6 Zika Virus Envelope Protein 0.0069 82.6 Dengue Virus 4 Envelope Protein 0.0041 49.2 HIV-1 Clade C GP120 0.0055 65.9 EBOV Envelope Glycoprotein 0.0106 126.1

Example 13: Binding of VP019, VP020, VP025-CT and VP025-F4 to Phosphatidyl Serine And viral antigens

[0363] The binding of VP019, VP020, VP025-CT (heterodimer mixture) and VP025-F4 (78% pure heterodimer) to a biotin-phosphatidyl serine and a select group of viral antigens (Influenza A H1N1 HA, Human RSV Glycoprotein G, Zika Virus Envelope Protein and SARS-Cov-2 S D614G) was investigated by ELISA. Binding curves are shown in FIGS. 10a and 10b and EC50 values are shown in Table 18 and Table 19. VP025-F4 had stronger binding to all of the antigens than VP025-CT.

TABLE-US-00022 EC50 in mg/mL for binding of VP019, VP020, VP025-CT and VP025-F4 to phosphatidyl serine and viral antigens VP019-F2 VP020 VP025-CT VP025-F4 Biotin-PS 0.0098 0.0296 0.0056 0.0018 Influenza A H1N1 HA 0.0021 0.0320 0.0034 0.0014 HRSV (A) Glycoprotein G 0.0073 0.0069 0.0043 0.0020 Zika Virus Envelope Protein 0.0010 0.0074 0.0020 0.0008 SARS-Cov-2 S D614G 0.0017 0.0045 0.0012 0.0008

TABLE-US-00023 EC50 in nM for binding of VP019, VP020, VP025-CT and VP025-F4 to phosphatidyl serine and viral antigens VP019-F2 VP020 VP025-CT VP025-F4 Biotin-PS 121.1 340.7 66.6 21.9 Influenza A H1N1 HA 25.8 368.8 40.6 17.0 HRSV (A) Glycoprotein G 89.9 79.6 51.4 23.6 Zika Virus Envelope Protein 12.2 85.1 24.0 10.0 SARS-Cov-2 S D614G 21.1 52.5 13.8 10.0

Example 14: Synthesis of Furin Inhibitor Linker-payloads

[0364] Solvents and reagents were purchased from Sigma-Aldrich, VWR, or Fisher Scientific, and used without further purification. Reactions were monitored either by thin-layer chromatography (TLC) or by analytical liquid chromatography-mass spectrometry (LC-MS) employing a Waters Acquity Ultra Performance LC system and a Synapt high-definition mass spectrometer. .sup.1H NMR spectra were recorded on a Varian Unity INOVA spectrometer (500 MHz). All chemical shifts are reported in ppm and coupling constants, J, are reported in hertz (Hz). NMR solvent peaks were referenced as follows: (.sup.1H NMR) CDCl.sub.3: 7.27 ppm, DMSO-d.sub.6: 2.50 ppm. Compounds were purified by flash column chromatography on a Teledyne ISCO Combi-Flash system using normal phase silica gel (SiliCycle Inc.) or reverse phase (Teledyne Gold- C18 or C18-Aq) pre-packed columns. The purity of compounds was determined by analytical HPLC (Waters Acquity Ultra Performance) using an Acquity UPLC CSH C18 1.7 .Math.m (50 mm x 2.1 mm) column and flow rate of 0.3 mL/min. Gradient conditions: solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile): 0-0.1 min 95% A, 0.1-4.0 min 5-95% B (linear gradient), 4.0-5.0 min 95% B, UV detection at 254 nm and 220 nm.

[0365] The reaction scheme is shown in FIG. 12.

[0366] N-methyl morpholine (13.8 .Math.lL, 0.126 mmol) was added to a solution of hexa-D-Arg (D-Argininamide D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-arginyl-D-alanine; Ambeed, cat# A333458) (30 mg, 0.0314 mmol), MC-Val-Cit-PAB-PNP (BroadPharm Cat#: BP-23292, CAS: 159857-81-5) (46.4 mg, 0.0629 mmol) and HOBt.H.sub.2O (1-hydroxybenzotriazole monohydrate; 5.3 mg, 0.0345) in anhydrous DMF (1 mL) under argon atmosphere. The solution was stirred at r.t. for 18 hours. The reaction was diluted with 1:1 ACN/water (0.05% HCO.sub.2H) (10 mL) and purified by reverse phase C18-Aq flash chromatography (gradient elution; 100% water -100% ACN with 0.05% HCO.sub.2H as mobile phase additive) to afford MC-VC-PAB-(D-Arg).sub.6-NH.sub.2 (12 mg, 0.00773 mmol, 25%) as a white solid after lyophilization. .sup.1H NMR (500 MHz, DMSO-d.sub.6) d 10.09 (s, 1H), 8.65 (br. s, 6H), 8.47 (s, 6H), 8.32 - 8.45 (m, 4H), 8.12 - 8.20 (m, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.50 - 7.80 (m, 18H), 7.29 (d, J = 7.8 Hz, 2H), 7.17 (s, 1H), 7.01 (s, 2H), 6.08 - 6.10 (m, 1H), 5.43 - 5.50 (s, 2H), 4.89 - 5.01 (m, 2H), 4.36 - 4.41 (m, 1H), 4.12 - 4.30 (m, 6H), 4.01 - 4.09 (m, 1H), 3.35 - 3.40 (m, 2H), 2.91- 3.12 (m, 12H), 2.09 - 2.21 (m, 2H), 1.93 - 2.00 (m, 1H), 1.65 -1.75 (m, 6H), 1.40 -1.62 (m, 28H), 1.30 -1.40 (m, 1H), 1.15 - 1.25 (m, 2H), 0.80 - 0.89 (m, 6H); MS (ESI) m/z: 1552.4 [M+H].sup.+.

[0367] The structure is shown in FIG. 11.

Example 15: Conjugation of Furin Inhibitor Payload to VP020

[0368] A test conjugation of Hexa-D-arginine linker-compound to VP020 was performed by reacting VP025 with 4 equivalents of TCEP and incubating at 37° C. for 1 hour to reduce the free cysteines. The sample was run through a Zeba column to remove TCEP and buffer exchanged into 1x PBS containing 1 mM DTPA pH 6.5. The sample was then reacted with 2.5 equivalents of the payload (Mc-VC-PAB-(D-Arg6)-CONH2 at Room temperature for 1 hour. The final product was analyzed by mass spectrometry (see FIG. 13) and shown to have the mass of 88,136 Da, near the expected mass of 88,138 Da.

Example 16: RSV Neutralization Assay for VP019, VP020 and VP025

[0369] A microneutralization assay was done to determine the antiviral properties of three compounds (VP019-F2, VP020 and VP025-F4) against RSV. Each virus was incubated with each antibody for 1 hour, after which the mix was added to A549 cells (human lung cancer cell line). Antiviral activity was determined 24 h later using an immunofluorescence-based assay. After 24 h, the infection plates were washed with PBS, fixed for 30 mins with 4% formaldehyde, washed again with PBS, and stored in PBS at 4° C. until staining. Any residual formaldehyde was quenched with 50 mM ammonium chloride, after which cells were permeabilized (0.1% Triton X100) and stained with an antibody recognizing RSV fusion protein (GeneTex GTX40697). The primary antibody was detected with an Alexa-488 conjugate secondary antibody (Life Technologies, A21244 and A11001), and nuclei were stained with Hoechst. Images were acquired on an Celllnsight CX5 high content platform (Thermo Scientific), and percentage infection calculated using CellInsight CX5 software (infected cells/total cells x 100).

[0370] The test articles were used in concentrations of 0.5 .Math.M and samples were tested in triplicate. The resulting data are shown in FIG. 14. For RSV, VP019-F2 had 97% viral inhibition, compared to 55% inhibition for VP025-F5 and 28% inhibition for VP020.

Example 17: ZIKV Neutralization Assay for VP025

[0371] A second neutralization assay was done for Zika virus using VP025-F4 using the following procedures.

Cell Culture Preparation

[0372] Vero E6 cells were maintained with DMEM supplemented with 2% FBS and (1% pen-strep -need to confirm with Allen) and stored at 37° C. with 5% CO.sub.2. Cells were seeded onto 48-well plates at a concentration of 8.0*10.sup.4 cells per well and allowed to adhere overnight. On the morning of infection cell monolayers were examined to ensure 90-95% confluency.

Test Zika Virus Neutralization

[0373] VP025-F4 was serially diluted in triplicate using infection media at a ratio of 1:3 for a total of eight dilutions (220 to 0.1 .Math.g/mL).). Zika virus (ZIKV), strain MEX-I-44, at a MOI of 1.0 (8.0*10.sup.4 FFU) was added to each dilution, mixed, and incubated at 37° C. and 5% CO.sub.2 for one hour.

Positive Control Neutralization

[0374] Simultaneous to the VP025:ZIKV incubation, positive control samples were also incubated. Mouse α-ZIKV MIAF (mouse immune ascitic fluid antibody) was diluted 1:500, 1:1000, and 1:1500 and combined with ZIKV, in triplicate, using the same concentration of virus as the test wells.

Plate Infection

[0375] Following incubation of VP025-F4:ZIKV and α-ZIKV MIAF:ZIKV, the Vero E6 well plate was removed from the incubator. Media was aspirated from the cells and the test and positive control samples were transferred to the Vero E6 plate and returned to the incubator to allow non-neutralized virus to infect cells for one hour.

Negative Control Focus Forming Assay

[0376] Simultaneous to the Plate Infection incubation, ZIKV was serially diluted (10.sup.-2 to 10.sup.-5) and samples were allowed to infect Vero E6 cells in triplicate.

Focus Reduction Neutralization Assay

[0377] Following the one hour incubation on Vero E6 cells an overlay of 0.8% methylcellulose was added to all wells and they were maintained at 37° C. and 5% CO.sub.2 for approximately 60 hours.

[0378] Plates were removed from the incubator, overlay was aspirated, and cells were gently washed twice with phosphate buffered saline. Virus was inactivated with a 1:1 fixative mixture of methanol and acetone which was allowed to fix plates for 30 minutes. Following inactivation, fixative was removed, and plates were allowed to air dry until no fixative remained.

[0379] All incubations and washes were performed at room temperature and plate was placed on a plate rocker. Cells were permeabilized with 0.5% Triton in PBS and washed with 0.02% Tween 20 in PBS (PBST). Blocking solution of PBST with BSA and normal goat serum was prepared and incubated on all wells for one hour. Primary antibody, mouse α-ZIKV MIAF, was diluted in PBST with BSA and stored on ice until used. Blocking solution was removed and 1° antibody was incubated for one hour. Antibody was removed and plates were washed with PBST. Secondary antibody, goat α-mouse IgG (high and low chain) HRP conjugated, was diluted in PBST with BSA. Antibody was incubated on wells for one hour and then wells were washed with PBST with BSA. Vector labs ImmPACT AMEC developing solution was prepared according to kit instructions and added to each well. Plate was incubated in the dark but checked regularly for staining. After foci were clearly developed (.sup.~15 minutes), wells were rinsed with deionized water and the plate was allowed to dry.

Results

[0380] No foci were observed in the VP025-F4 test wells or the positive control wells (see FIG. 14b). Foci developed as expected in the negative control focus-forming assay wells demonstrating decreasing foci with each successive virus dilution. This indicates that the VP025 test article was able to neutralize ZIKV at dilutions from (220 to 0.1 .Math.g/mL).