Vaccine Compositions and Antibodies For Lyme Disease

Abstract

The present invention relates to vaccine compositions comprising lipid antigens, antibodies targeting lipid antigens, pharmaceutical compositions comprising such and their use in diagnosing, monitoring, treating, and preventing infectious disease, such as Lyme disease. In one aspect, administered is a therapeutically effective amount of a vaccine composition comprising a lipid antigen, an antibody or fragment thereof binding a lipid antigen, and/or a pharmaceutical composition comprising an antibody or fragment thereof binding a lipid antigen. Other aspects are described.

Claims

1. A method of preventing, treating and/or alleviating an infectious disease comprising administering to a patient in need thereof a therapeutically effective amount of a. a vaccine composition comprising a lipid antigen, b. an antibody or fragment thereof binding a lipid antigen, and/or c. a pharmaceutical composition comprising an antibody or fragment thereof binding a lipid antigen.

2. A method of preventing, treating and/or alleviating Lyme disease comprising administering to a patient in need thereof a therapeutically effective amount of a. a vaccine composition comprising a lipid antigen, b. an antibody or fragment thereof binding a lipid antigen, and/or c. a pharmaceutical composition comprising an antibody or fragment thereof binding a lipid antigen.

3. A method for producing an antibody or fragment thereof binding a lipid antigen comprising (i) Providing a vaccine composition comprising a lipid antigen, (ii) Administering said vaccine composition to an animal or human, to trigger or promote the production of an antibody or fragment thereof binding to said lipid antigen, (iii) Obtaining said antibody or fragment thereof binding a lipid antigen from said animal or human.

4. A vaccine composition comprising a lipid antigen.

5. The vaccine composition according to claim 4, wherein said lipid antigen is a glycolipid antigen.

6. The vaccine composition according to any of the preceding claims, wherein said lipid antigen is a BBGL-1 and/or BBGL-2 antigen.

7. The vaccine composition according to any of the preceding claims, wherein said antigen is synthetically produced.

8. The vaccine composition according to any of the preceding claims, wherein said antigen is encapsulated in a vesicle comprising at least one lipid layer.

9. The vaccine composition according to any of the preceding claims, wherein said vesicle comprises at least one lipid bilayer.

10. The vaccine composition according to any of the preceding claims, wherein said vesicle is selected from the group consisting of solid lipid nanoparticles (SLNs), emulsions, liposomes, micelles, and bilayer sheets.

11. The vaccine composition according to any of the preceding claims, wherein said vesicle comprises a lipid component selected from the group consisting of phosphatidylcholine, phosphatidylglycerol and cholesterol.

12. The vaccine composition according to any of the preceding claims, wherein said antigen is conjugated to a carrier protein.

13. The vaccine composition according to any of the preceding claims, wherein said antigen is covalently conjugated to a carrier protein.

14. The vaccine composition according to any of the preceding claims, wherein said carrier protein is an immunogenic carrier protein.

15. The vaccine composition according to any of the preceding claims, wherein said carrier protein is selected from the group consisting of bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH).

16. The vaccine composition according to any of the preceding claims, wherein said vaccine is a multivalent vaccine.

17. The vaccine composition according to any of the preceding claims further comprising one or more pharmaceutically acceptable excipients.

18. The vaccine composition according to any of the preceding claims further comprising an adjuvant.

19. The vaccine composition according to any of the preceding claims, wherein said adjuvant is β-glucan.

20. The vaccine composition according to any of the preceding claims, wherein said vaccine is for an administration form selected among subcutaneous, intradermal, intramuscular, intravenous, oral and nasal.

21. The vaccine composition according to any of the preceding claims, wherein said vaccine is used for prevention, treatment and/or alleviation of Lyme disease.

22. The vaccine composition according to any of the preceding claims, wherein said vaccine is for prevention, treatment and/or alleviation of a condition related to and/or caused by Lyme disease.

23. The vaccine composition according to any of the preceding claims, wherein said condition is selected among Lyme Borreliosis, antibiotic resistant Lyme Borreliosis, synovitis, arthritis, antibiotic refractory arthritis, fatigue, sleep impairment, nerve pain, headaches, memory loss, joint pain and/or depression.

24. The vaccine composition according to any of the preceding claims, comprising one or more liposome(s) comprising one or more lipid antigen(s) embedded in the outer membrane of said liposome(s).

25. The vaccine composition according to claim 24, wherein the one or more lipid antigens are selected among BBGL1, BBGL2 and combinations thereof.

26. The vaccine composition according to claim 24 or 25, wherein the lipids of the liposome comprises one or more of phosphatidylserine (PS), 1,2-dioleyl- sn-glycero-3-phospho-L-serine (DOPS), distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), DSPE-PEG2000, distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DSPG), di-(9Z-octadecanoyl-sn-glycero-3-phosphocholine (DOPC), di-hexadecanoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin, di-oleyl-sn-glycero-3-phosphoethanilamine (DOPE) and cholesterol.

27. The vaccine composition according to any of claims 24 - 26, wherein the lipids of the liposome comprise DSPE, DSPG and cholesterol.

28. The vaccine composition according to claim 27, wherein the lipids of the liposome consist of DSPE, DSPG and cholesterol.

29. The vaccine composition according to claim 28, wherein the composition of the liposomes is selected among: DSPC/DSPG/Cholesterol/BBGL1 in the weight ratio: 7:2:1:1; DSPC/DSPG/Cholesterol/BBGL2 in the weight ratio: 7:2:1:1; and DSPC/DSPG/Cholesterol/BBGL1/BBGL2 in the weight ratio: 7:2:1:1:1.

30. The vaccine composition according to any of the claims 24-29, wherein the liposome further comprises a vaccine adjuvant and/or immunostimulant encapsuled inside the liposomes.

31. A method for prevention, treatment and/or alleviation of Lyme disease by administering the vaccine according to any of the preceding claims to a subject in need thereof.

32. The method according to claim 31, wherein said vaccine is administered in an administration form selected among subcutaneous, intradermal, intramuscular, intravenous, oral and nasal.

33. The method according to claim 31, wherein said vaccine is administered in an administration form selected among oral and nasal.

34. A compound selected among 6-[12-(X-amino)-dodecanoyl]-1-cholesteryl-β-D-galactopyranoside, where X is an amino protective group; Allyl 2,3,4,6-tetra-O-X1-α-D-galactopyranoside, (2′R)-2′,3′-Epoxypropyl 2,3,4,6-tetra-O-X1-α-D-galactopyranoside, (2′R)-3′-Bromo-2′-hydroxypropyl 2,3,4,6-tetra-O-X1-α-D-galactopyranoside, (2′R)-3′-Bromo-2′-palmitoyloxypropyl 2,3,4,6-tetra-O-X1-α-D-galactopyranoside, 1-O-oleoyl-2-O-palmitoyl-sn-glyceryl 2,3,4,6-tetra-O-X1-α-D-galactopyranoside,wherein X1 is an alcohol protection group; and (2′R)-3′-Bromo-2′-[12-(X2-amino)-dodecanoyl]-2,3,4,6-tetra-O-X3-α-D-galactopyranoside, (2′R)-3′-Bromo-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-OX3-α-D-galactopyranoside, (2′R)-3′-oleoyl-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-X3-a-D-galactopyranoside, wherein X2 is an amine protective group and X3 is an alcohol protective group.

35. The compound of claim 34 selected among: 6-[12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoyl]-1-cholesteryl-β-D-galactopyranoside (7), Allyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (9), (2′R)-2′,3′-Epoxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (10), (2′R)-3′-Bromo-2′-hydroxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (11) (2′R)-3′-Bromo-2′-palmitoyloxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (12), 1-O-oleoyl-2-O-palmitoyl-sn-glyceryl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (13), (2′R)-3′-Bromo-2′-[12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (14), (2′R)-3′-Bromo-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (15) and (2′R)-3′-oleoyl-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (16).

36. A compound selected among BBGL-1-Biotin and BBGL-2-Biotin.

37. An antibody or fragment thereof binding a lipid antigen.

38. The antibody or fragment thereof according to claim 37, wherein said lipid antigen is a glycolipid antigen.

39. The antibody or fragment thereof according to any of claims 37-38, wherein said lipid antigen is a BBGL-1 and/or BBGL-2 antigen.

40. The antibody or fragment thereof according to any of claims 37-39 binding a BBGL-1 antigen.

41. The antibody or fragment thereof according to any of claims 37-40 binding a BBGL-2 antigen.

42. The antibody or fragment thereof according to any of claims 37-41, binding a BBGL-1 antigen and a BBGL-2 antigen.

43. The antibody or fragment thereof according to any of claims 37-42, wherein said antibody or fragment thereof comprises a homodimeric immunoglobulin or fragment thereof.

44. The antibody or fragment thereof according to any of claims 37-43, wherein said antibody or fragment thereof comprises a heterodimeric immunoglobulin or fragment thereof.

45. The antibody or fragment thereof according to any of claims 37-44, wherein said antibody or fragment thereof is an IgG, IgM IgA, IgD and/or an IgE.

46. The antibody or fragment thereof according to any of claims 37-45, wherein said antibody or fragment thereof is an IgG1, IgG3 and/or IgM.

47. The antibody or fragment thereof according to any of claims 37-46, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs 1-20.

48. The antibody or fragment thereof according to any of claims 37-47, wherein said antibody or fragment thereof comprises a sequence combination selected from the specific combinations given in Table 3.

49. A method of producing an antibody or fragment thereof according to any of claims 37-48, wherein said antibody or fragment thereof is produced by animal immunization.

50. The method according to claim 49, wherein said animal immunization comprises administering a vaccine according to any of the precedent claims to an animal.

51. The method according to any of claims 49-50, wherein said animal is selected from the group consisting of mouse, rabbit, alpaca and llama.

52. A method of producing an antibody or fragment thereof according to any of claims 49-51, comprising a. Isolating B cells from a Lyme diseases patient, b. Isolating B-cell receptors from said B cells, c. Sequencing said B-cell receptors, and d. Cloning said B-cell receptors.

53. A method of producing an antibody or fragment thereof according to any of claims 49-52, using a human antibody phage display library.

54. An antibody or fragment thereof obtainable by the method of any of the claims 49-53.

55. The antibody or fragment thereof according to any of claims 37-48, wherein said antibody or fragment thereof further binds a CD3 antigen.

56. The antibody or fragment thereof according to any of claims 37-48 and 55, wherein said antibody or fragment thereof is a T cell engaging antibody.

57. The antibody or fragment thereof according to any of claims 37-48 and 55-56, wherein said antibody or fragment thereof comprises variable light chain CDR sequences of SEQ ID NOs:75-77.

58. The antibody or fragment thereof according to any of claims 37-48 and 55-57, wherein said antibody or fragment thereof comprises variable heavy chain CDR sequences of SEQ ID NOs:78-80.

59. The antibody or fragment thereof according to any of claims 37-48 and 55-58, wherein said antibody or fragment thereof comprises variable light chain CDR sequences of SEQ ID NOs:75-77 and variable heavy chain CDR sequences of SEQ ID NOs:78-80.

60. The antibody or fragment thereof according to any of claims 37-48 and 55-59, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs 21-38.

61. The antibody or fragment thereof according to any of claims 37-48 and 55-60, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs 39-57.

62. The antibody or fragment thereof according to any of claims 37-48 and 55-61, wherein said antibody or fragment thereof comprises a sequence combination selected from the specific combinations given in Table 4.

63. The antibody or fragment thereof according to any of claims 37-48 and 55-62, wherein said antibody or fragment thereof further binds a TCR Vα24Jα18 antigen.

64. The antibody or fragment thereof according to any of claims 37-48 and 55-63, wherein said TCR Vα24Jα18 antigen comprises a sequence according to SEQ ID No.: 81, 82 and/or 83.

65. The antibody or fragment thereof according to any of claims 37-48 and 55-64, wherein said antibody is an NKT cell engaging antibody.

66. The antibody or fragment thereof according to any of claims 37-48 and 55-65, wherein said antibody or fragment thereof comprises a variable light chain sequence selected from the group consisting of SEQ ID NOs 58-60.

67. The antibody or fragment thereof according to any of claims 37-48 and 55-66, wherein said antibody or fragment thereof comprises a variable heavy chain sequence selected from the group consisting of SEQ ID NOs 61-63.

68. The antibody or fragment thereof according to any of claims 37-48 and 55-67, wherein said antibody or fragment thereof comprises a variable light chain sequence selected from the group consisting of SEQ ID NOs 58-60 and a variable heavy chain sequence selected from the group consisting of SEQ ID NOs 61-63.

69. The antibody or fragment thereof according to any of claims 37-48 and 55-68, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs 64-69.

70. The antibody or fragment thereof according to any of claims 37-48 and 55-69, wherein said antibody or fragment thereof comprises a sequence combination selected from the specific combinations given in Table 10.

71. The antibody or fragment thereof according to any of claims 37-48 and 55-70, wherein said antibody or fragment thereof further binds a CD16 and/or a CD56 antigen.

72. The antibody or fragment thereof according to any of claims 37-48 and 55-71, wherein said antibody or fragment thereof further binds a CD16 and a CD56 antigen.

73. The antibody or fragment thereof according to any of claims 37-48 and 55-72, wherein said antibody or fragment thereof specifically activates NKT cells.

74. The antibody or fragment thereof according to any of claims 37-48 and 55-73, wherein said antibody or fragment thereof comprises a sequence combination selected from the specific combinations given in Table 11.

75. The antibody or fragment thereof according to any of claims 37-48 and 55-74, wherein said antibody or fragment thereof engage a cell selected from the group consisting of T cells, NK cells and NKT cells.

76. The antibody or fragment thereof according to any of claims 37-48 and 55-75, wherein said antibody or fragment thereof further comprises a drug payload.

77. The antibody or fragment thereof according to any of claims 37-48 and 55-76, wherein said drug payload is an infrared dye and/or a radionucleotide.

78. The antibody or fragment thereof according to any of claims 37-48 and 55-77, wherein said antibody or fragment thereof comprises a mutation selected from the group consisting of an Ala339Cys, a Ser337Cys and a Lys340Cys mutation.

79. The antibody or fragment thereof according to any of claims 37-48 and 55-77, wherein said antibody or fragment thereof comprises an Ala339Cys mutation.

80. The antibody or fragment thereof according to any of claims 37-48 and 55-79, wherein said drug payload is inserted at a site-specific free Cys residue.

81. The antibody or fragment thereof according to any of claims 37-48 and 55-80, wherein said antibody or fragment thereof comprises a sequence selected from the group consisting of SEQ ID NOs 70-73.

82. The antibody or fragment thereof according to any of claims 37-48 and 55-81, wherein said antibody or fragment thereof comprises a sequence combination selected from the specific combinations given in Table 13.

83. The antibody or fragment thereof according to any of claims 37-48 and 55-82, wherein said antibody or fragment thereof is a scFv.

84. The antibody or fragment thereof according to any of claims 37-48 and 55-83, wherein said antibody or fragment thereof comprises a scFv.

85. The antibody or fragment thereof according to any of claims 37-48 and 55-84, wherein said antibody or fragment thereof provides enhanced ADCC, ADCP and/or CDC.

86. The antibody or fragment thereof according to any of claims 37-48 and 55-85, comprising a first antigen binding site capable of binding to a first antigen and a second antigen binding site capable of binding to a second antigen, wherein said first antigen binding site is comprised in a Fab fragment and said second antigen binding site is comprised in a moiety selected from the group consisting of scFv, antibody fragment and protein moiety, characterized by said moiety being attached to the light chain of said Fab fragment.

87. The antibody or fragment thereof according to any of claim 37-48 and 55-86, wherein said moiety is a scFv.

88. The antibody or fragment thereof according to any of claim 37-48 and 55-87, wherein said moiety is attached to the C-terminus or N-terminus of the light chain of said Fab fragment.

89. The antibody or fragment thereof according to any of claim 37-48 and 55-88, wherein said moiety is attached to the C-terminus of the light chain of said Fab fragment.

90. The antibody or fragment thereof according to any of claim 37-48 and 55-89, wherein the constant region is derived from an IgG or an IgM.

91. The antibody or fragment thereof according to any of claim 37-48 and 55-90, wherein said antibody or fragment thereof comprises a mutation in the IgM constant region, preferably at position 131, 135, 354 and/or 385.

92. The antibody or fragment thereof according to any of claims 37-48 and 55-91, wherein said antibody or fragment thereof comprises a mutation in the IgM constant region selected from the group consisting of K131R, K131H, K131D, K131E, Q135K, Q135R, Q135H, Q135D, Q135E, T354D, T354E, T354K, T354R, T354H, E385D, E385K, E385R and E385H.

93. The antibody or fragment thereof according to any of claims 37-48 and 55-92, wherein said Fab fragment is derived from an IgG.

94. The antibody or fragment thereof according to any of claim 37-48 and 55-93, wherein said Fab fragment is derived from an IgG selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

95. The antibody or fragment thereof according to any of claim 37-48 and 55-94, wherein said Fab fragment is derived from an IgG2 or an IgG4.

96. The antibody or fragment thereof according to any of claim 37-48 and 55-95, wherein said antibody or fragment thereof comprises a null fc.

97. The antibody or fragment thereof according to any of claim 37-48 and 55-96, wherein said antibody or fragment thereof comprises a null fc, wherein said null fc comprises a Leu234Ala and/or a Leu235Ala and/or a Lys22Ala mutation.

98. The antibody or fragment thereof according to any of claim 37-48 and 55-97, wherein said antibody or fragment thereof comprises a Ser228Pro mutation.

99. The antibody or fragment thereof according to any of claim 37-48 and 55-98, comprising a linker.

100. The antibody or fragment thereof according to any of claim 37-48 and 55-99, wherein said linker is a peptide linker.

101. The antibody or fragment thereof according to any of claim 37-48 and 55-100, wherein said linker is a GlySer linker.

102. The antibody or fragment thereof according to any of claims 37-48 and 55-101, wherein said linker comprises the sequence of SEQ ID NO:71.

103. The antibody or fragment thereof according to any of claim 37-48 and 55-102, wherein said antibody or fragment thereof blocks host cell entry of a pathogen.

104. A pharmaceutical composition comprising an antibody or fragment thereof according to any of claim 37-48 and 55-103, optionally comprising one or more excipients such as diluents, binders or carriers.

105. The pharmaceutical composition according to claim 104, further comprising an adjuvant.

106. The pharmaceutical composition according to claim 105, wherein said adjuvant is β-glucan.

107. The antibody or fragment thereof or pharmaceutical composition according to any of the preceding claims, wherein said antibody or fragment thereof or pharmaceutical composition is for an administration form selected among subcutaneous, intradermal, intramuscular, intravenous, oral and nasal.

108. An isolated nucleic acid molecule encoding an antibody or fragment thereof according to any of the precedent claims.

109. A recombinant vector comprising the nucleic acid molecule of claim 108.

110. A host cell comprising the recombinant vector of claim 109.

111. A method for the production of an antibody or fragment thereof according to any of the precedent claims comprising a step of culturing the host cell according to claim 110, in a culture medium under conditions allowing the expression of the antibody or fragment thereof and separating the antibody or fragment thereof from the culture medium.

112. A method for the production of an antibody or fragment thereof according toany of the precedent claims comprising a synthetic and/or recombinant step.

113. Use of an antibody or fragment thereof or vaccine composition according to any of the preceding claims in a preclinical model.

114. A method of preventing, treating and/or alleviating an infectious disease comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof or pharmaceutical composition according to any of the preceding claims.

115. The method according to any of the preceding claims, wherein said infectious disease is caused by a Borrelia Burgdorferi bacteria sensu lato.

116. The method according to any of the preceding claims, wherein said Borrelia Burgdorferi bacteria sensu lato is B. burgdorferi, B. mayonii, B. afzelii and/or B. garinii.

117. A method of preventing, treating and/or alleviating a condition related to and/or caused by Lyme disease comprising administration to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof or pharmaceutical composition according to any of the preceding claims.

118. The method according to any of the preceding claims, wherein the condition is selected among Lyme Borreliosis, antibiotic resistant Lyme Borreliosis, synovitis, arthritis, antibiotic refractory arthritis, fatigue, sleep impairment, nerve pain, headaches, memory loss, joint pain and/or depression.

119. A method of preventing, treating and/or alleviating Lyme disease comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof or pharmaceutical composition according to any of the preceding claims.

120. A method of diagnosing Lyme disease comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof or pharmaceutical composition according to any of the preceding claims.

121. A method of diagnosing Lyme disease comprising the steps of a. obtaining a blood or urine sample from a subject, and b. testing said blood or urine sample for the presence of a BBGL-1 and/or BBGL-2 biomarker with the antibody or fragment thereof according to any of the preceding claims.

122. A method of selecting and/or identifying a subject that might benefit from treatment with an antibody or fragment thereof or vaccine according to any of the preceding claims comprising the steps of a. obtaining a blood or urine sample from a subject, and b. testing said blood or urine sample for the presence of a BBGL-1 and/or BBGL-2 biomarker with the antibody or fragment thereof according to any of the preceding claims.

123. A method of monitoring infectious disease progression and/or disease resolution comprising the steps of a. obtaining a blood or urine sample from a subject, and b. testing said blood or urine sample for the presence of a BBGL-1 and/or BBGL-2 biomarker with the antibody or fragment thereof according to any of the preceding claims.

124. A method of detecting BBGL1 and/or BBGL2 specific antibodies in a blood sample using BBGL1-biotin and/or BBGL2-Biotin.

125. A method of in vivo imaging of bacterial infections comprising use of an antibody or fragment thereof according to any of the precedent claims.

126. The antibody or fragment thereof according to any of the precedent claims for use in an imaging technique for monitoring infectious disease progression/resolution.

127. A diagnostic kit comprising an antibody or fragment thereof according to any of the precedent claims and instructions for use.

128. A kit for screening comprising an antibody or fragment thereof according to any of the precedent claims and instructions for use.

129. An antibody or fragment thereof, preferably according to any of the preceding claims, capable of binding a TCR Vα24Jα18 antigen, wherein said antibody or fragment thereof is capable of binding an additional antigen, such as a lipid antigen, allowing NKT cells to be re-directed to said additional antigen.

130. An antibody or fragment thereof, preferably according to any of the preceding claims, capable of binding a CD3 antigen, and wherein said antibody or fragment further is capable of binding a CD16 and/or a CD56 antigen; and preferably wherein said antibody or fragment thereof is capable of binding an additional antigen, such as a lipid antigen, allowing NKT cells to be re-directed to said additional antigen.

131. An antibody or fragment thereof, preferably according to any of the preceding claims, capable of binding a TCR Vα24Jα18 antigen, wherein said antibody is an NKT cell engaging antibody, wherein said antibody or fragment thereof further is capable of binding CD3 antigen, and wherein said antibody or fragment thereof further is capable of binding a CD16 and/or a CD56 antigen.

132. The antibody or fragment thereof according to any of the preceding claims, wherein said TCR Vα24Jα18 antigen comprises a sequence according to SEQ ID NO.: 81, 82 and/or 83.

133. The antibody or fragment thereof according to any of the preceding claims, wherein said antibody or fragment thereof comprises a. a variable light chain sequence comprising at least one of the CDR regions according to SEQ ID No.: 84 - 86; and/or b. A variable heavy chain sequence comprising at least one of the CDR regions according to SEQ ID No.: 87 - 89.

134. The antibody or fragment thereof according to any of the preceding claims, wherein said CD3 antigen and said CD16 and/or a CD56 antigen are expressed on NKT cells.

135. The antibody or fragment thereof according to any of the preceding claims, wherein said antibody or fragment thereof is capable of binding to said CD3 antigen and simultaneously to said CD16 and/or CD56 antigen.

Description

FIGURES

[0290] FIG. 1 shows two Lyme Disease antigens BBGL-1 (cholesteryl 6-O-palmitoyl-β-D-galactopyranoside) and BBGL-2 (1-oleoyl-2-palmitoyl-3-(α-D-galactosyl)-sn-glycerol).

[0291] FIG. 2 shows three vaccine constructs containing either anti-BBGL-1, anti-BBGL-2 or the combination of anti-BBGL-1 and anti-BBGL-2.

[0292] FIG. 3 shows anti-BBGL-1 and anti-BBGL-2 antibodies.

[0293] FIG. 4 shows anti-BBGL-1 and anti-BBGL-2 bispecific antibodies with T cell engager effector functions.

[0294] FIG. 5 shows anti-BBGL-1 and anti-BBGL-2 bispecific antibodies with NKT cell engager effector functions.

[0295] FIG. 6 shows anti-BBGL-1 and anti-BBGL-2 multi-specific antibodies with T cell, NK cell, and NKT cell engager effector functions.

[0296] FIG. 7a shows in vitro neutralization of Borrelia burgdorferi of Day 30 serum samples from immunized mice (n = 6 mice per group)

[0297] FIG. 7b shows the average weight of mice (in grams with standard deviation) per group of immunized mice (12 mice per group) over the 60 day experiment

[0298] FIG. 8 Synthesis of BbGL1

[0299] FIG. 9 Synthesis of BbGL1-Biotin

[0300] FIG. 10 Synthesis of BbGL2

[0301] FIG. 11 Synthesis of BbGL2-Biotin

[0302] All cited references are incorporated by reference.

[0303] 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.

[0304] 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: Design of BBGL-1 and BBGL-2 Vaccines

[0305] Three vaccine constructs were designed that contain either BBGL-1, BBGL-2 or the combination of BBGL-1 and BBGL-2 (see FIG. 2).

[0306] In format VIT-200, BBGL-1 and/or BBGL-2 are reconstituted into liposomes, solid lipid nanoparticles or micelles containing other lipid components, such as phosphatidylcholine, phosphatidylglycerol and cholesterol.

[0307] In format VIT-201, BBGL-1 and/or BBGL-2 are covalently conjugated to immunogenic carrier proteins such as BSA (bovine serum albumin) and KLH (keyhole limpet hemocyanin).

[0308] In format VIT-202, BBGL-1 and/or BBGL-2 are loaded onto the extracellular domain of recombinant human CD1d and human B2M (Beta-2-microglobulin). This allows for BBGL-1 and/or BBGL-2 to be directly presented to NKT cells.

Example 2: Design of anti-BBGL-1 and anti-BBGL-2 Antibodies with IgG1, IgG3 or IgM Effector Functions

[0309] Anti-BBGL-1 and anti-BBGL-2 antibodies are derived from immunization of animals (mouse, rabbit, llama) with vaccines from Example 1, or from B cell isolation and sequencing from Lyme disease patients.

[0310] In constructs VIT-203 and VIT-204 (FIG. 3), the anti-BBGL-1 and anti-BBGL-2 antibodies have homodimeric IgG1, IgG3 or IgM effector functions. In construct VIT-205 (FIG. 3), the anti-BBGL-1 x anti-BBGL-2 antibodies have heterodimeric IgG1, IgG3 or IgM effector functions.

[0311] In the below tables, the anti-BBGL1 antibodies have the VH sequences denoted as BBGL1-VH, VL-kappa sequences as BBGL1-VLK, and VL-lambda sequences as BBGL1-VLL. The anti-BBGL2 antibodies have the VH sequences denoted as BBGL2-VH, VL-kappa sequences as BBGL2-VLK, and VL-lambda sequences as BBGL2-VLL. The scFv versions of the above constructs will be termed BBGL1-VH-VLK-scFv, BBGL1-VH-VLL-scFv, BBGL2-VH-VKL-scFv, and BBGL2-VH-VLL-scFv.

TABLE-US-00001 Constant regions for IgG1-like constructs Construct Sequence Notes WT huIgG1 CH1-CH2-CH3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:1) V-IGG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:2) Same as above, hinge region underlined V-IGG1-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:3) Same as above, but with linker and fc only V-IGG1-A ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:4) K409R V-IGG1-A-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY Same as SEQ NO 4, but with linker and fc only SRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:5) V-IGG1-B ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO:6) F405L V-IGG1-B-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:7) Same as SEQ NO 6, but with linker and fc only

TABLE-US-00002 Constant regions for IgG3-like constructs Construct Sequence Notes WT huIgG3 CH1-CH2-CH3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTPLGDTTHTPEPKSCDTPPPCPR CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPEN NYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVM HEALHNRFTQKSLSLSPGK (SEQ ID NO:8) Hinge region underlined V-IGG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:9) Hinge region underlined with IgG4 hinge S228P mutation in bold V-IGG3-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:10) Same as SEQ NO 9, but with linker and fc only V-IGG3-A ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:11) K409R V-IGG3-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFL YSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:12) Same as SEQ NO 11, but with linker and fc only V-IGG3-B ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVESKYGPPCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:13) F405L V-IGG3-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLL YSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K (SEQ ID NO:14) Same as SEQ NO 13, but with linker and fc only V-IGG3-C ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV Utilizes an IgG1-like hinge FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK (SEQ ID NO:15) V-IGG3-C-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK (SEQ ID NO:16) Same as SEQ NO 15, but with linker and fc only V-IGG3-D ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK (SEQ ID NO:17) K409R V-IGG3-D-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFFLYSRLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK (SEQ ID NO:18) Same as SEQ NO 17, but with linker and fc only V-IGG3-E ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTC NVNHKPSNTKVDKRVELKTGDTTHTCPRCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVD GVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSD GSFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSL SLSPGK (SEQ ID NO:19) F405L V-IGG3-E-Fc GGGGSGGGGSGGGGSKTGDTTHTCPRCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG VEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ Same as SEQ NO 19, but with linker and fc only VSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDG SFLLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSL SPGK (SEQ ID NO:20)

TABLE-US-00003 Sequences of anti-BBGL1 and anti-BBGL-2 constructs with enhanced ADCC, ADCP and CDC. CLK = kappa CL domain, CLL = lambda CL domain Construct Sequence of Chain 1 Sequence of Chain 2 Sequence of Chain 3 Sequence of Chain 4 VIT-203 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) Same as Chain 1 (BBGL1-VH) - (V-IGG1 or V-IGG3 or V-IGG3-C) Same as Chain 3 VIT-204 (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) Same as Chain 1 (BBGL2-VH) - (V-IGG1 or V-IGG3 or V-IGG3-C) Same as Chain 3 VIT-205 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) (BBGL1-VH) - (V-IGG1-A or V-IGG3-A or V-IGG3-D) (BBGL2-VH)-(V-IGG1-B or V-IGG3-B or V-IGG3-E) VIT-203sc (BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) - (V-IGG1-Fc or V-IGG3-Fc or V-IGG3-C-Fc) Same as Chain 1 VIT-204sc (BBGL2-VH-VLK-scFv or BBGL2-VH-VLL-scFv)- (V-IGG1-Fc or V-IGG3-Fc or V-IGG3-C-Fc) Same as Chain 1 VIT-205sc (BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) - (V-IGG1-A-Fc or V-IGG3-A-Fc or V-IGG3-D-Fc) (BBGL2-VH-VLK-scFv or BBGL2-VH-VLL-scFv)- (V-IGG1-B-Fc or V-IGG3-B-Fc or V-IGG3-E-Fc)

Example 3: Design of Anti-BBGL-1 and Anti-BBGL-2 Antibodies with T Cell Engager Effector Functions

[0312] T cell engaging bispecific antibodies were designed by fusing a GlySer linker (GGGGSGGGGSGGGGS (=3xSEQ ID NO: 92)) and anti-CD3 scFv to C-terminus of the light chain of the anti-BBGL-1/BBGL-2 Fab domain (see FIG. 4). Null Fc fragments are utilized using IgG4 or IgG2 Fc region.

TABLE-US-00004 Sequences of anti-BBGL1 and anti-BBGL-2 constructs with T cell effector function. CLK = kappa CL domain, CLL = lambda CL domain Construct Sequence of Chain 1 Sequence of Chain 2 Sequence of Chain 3 Sequence of Chain 4 VIT-206 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(any scFv from Table 5) Same as Chain 1 (BBGL1-VH)-(V-IGG4-A or V-IGG2 or V-IGG2-C) Same as Chain 3 VIT-207 (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(any scFv from Table 5) Same as Chain 1 (BBGL2-VH)-(V-IGG4-A or V-IGG2 or V-IGG2-C) Same as Chain 3 VIT-208 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(any scFv from Table 5) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(any scFv from Table 5) (BBGL1-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (BBGL2-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT-209 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X (BBGL1-VH)- (BBGL2-VH)- SEQ ID NO: 92)-(any scFv from Table 5) (V-IGG4-A or V-IGG2-A or V-IGG2-D) (V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT210 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(any scFv from Table 5) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) (BBGL1-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (BBGL2-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E)

TABLE-US-00005 Anti-CD3 scFv sequences Construct Sequence VCD3-H1L1 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:21) VCD3-H1L1-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:22) VCD3-H1L2 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:23) VCD3-H1L2-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:24) VCD3-H1L3 QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKGLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:25) VCD3-H1L3-DS QVQLVQSGGGVVQPGRSLRLSCAASGYTFTRYTMHWVRQAPGKCLEWVGYINPSRGYTNYND SVKGRFTISTDKSKNTAYLQMNSLRAEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:26) VCD3-H2L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:27) VCD3-H2L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:28) VCD3-H2L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:29) VCD3-H2L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:30) VCD3-H2L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:31) VCD3-H2L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:32) VCD3-H3L1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:33) VCD3-H3L1-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCQASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFSGS GSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:34) VCD3-H3L2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:35) VCD3-H3L2-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRLIYDTSKRATGIPARFSGSGS GTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:36) VCD3-H3L3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQGLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGQGTKLEIK (SEQ ID NO:37) VCD3-H3L3-DS QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQSPGQCLEWMGYINPSRGYTNYN QKFQGRVTMTTDKSTSTAYMELSSLRSEDTAVYYCARYYDDHYSLDYWGQGTTVTVSSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGS EIQLTQSPATLSLSPGERATLSCRASSSVSYMNWYQQKPGQAPRRWIYDTSKLATGIPARFSGSG SGTDYTLTISSLEPEDAAVYYCQQWSSNPFTFGCGTKLEIK (SEQ ID NO:38)

TABLE-US-00006 Sequences for null Fc domains and heterodimerization Construct Sequence Notes WT huIgG1 CH1-CH2-CH3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:39) WT huIgG4 CH1-CH2-CH3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK (SEQ ID NO:40) V-IGG4-A ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK (SEQ ID NO:41) S228P FALA (F234A, L235A) K322A Naturally contains F405 and R409 V-IGG4-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:42) Same as above, but with linker and fc only V-IGG4-B ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK (SEQ ID NO:43) S228P FALA (F234A, L235A) K322A F405L, R409K V-IGG4-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:44) Same as above, but with linker and fc only WT huIgG2 CH1-CH2-CH3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWES NGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:45) V-IGG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWE SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:46) Contains IgG1 hinge (DKTHTCPPCPAP) VA (V234A) K322A V-IGG2-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPPAAGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIS VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:47) Same as above, but with linker and fc only V-IGG2-A ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWE SNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:48) Contains IgG1 hinge (DKTHTCPPCPAP) VA (V234A) K322A K409R V-IGG2-A-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPPAAGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIS VEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:49) Same as above, but with linker and fc only V-IGG2-B ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKDKTHTCPPCPAPPAAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWE SNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:50) Contains IgG1 hinge (DKTHTCPPCPAP) FALA (F234A) K322A F405L V-IGG2-B-Fc GGGGSGGGGSGGGGSDKTHTCPPCPAPPAAGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIS VEWESNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51) Same as above, but with linker and fc only V-IGG2-C ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKYGPPCPPCPAPPAAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWES NGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:52) Contains IgG4 hinge (YGPPCPPCPAP) with S228P FALA (V234A) K322A V-IGG2-C-Fc GGGGSGGGGSGGGGSYGPPCPPCPAPPAAGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISV EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:53) Same as above, but with linker and fc only V-IGG2-D ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKYGPPCPPCPAPPAAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWES NGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:54) Contains IgG4 hinge (YGPPCPPCPAP) with S228P VA (V234A) K322A K409R V-IGG2-D-Fc GGGGSGGGGSGGGGSYGPPCPPCPAPPAAGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISV EWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:55) Same as above, but with linker and fc only V-IGG2-E ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHK PSNTKVDKTVERKYGPPCPPCPAPPAAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWES NGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:56) Contains IgG4 hinge (YGPPCPPCPAP) with S228P FALA (FV34A) K322A F405L V-IGG2-E-Fc GGGGSGGGGSGGGGSYGPPCPPCPAPPAAGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCAVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISV EWESNGQPENNYKTTPPMLDSDGSFLLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:57) Same as above, but with linker and fc only

Example 4: Design of Anti-BBGL-1 and Anti-BBGL-2 Antibodies with NKT Cell Engager Effector Functions

[0313] NKT cell engaging bispecific antibodies were designed by fusing a GlySer linker (GGGGSGGGGSGGGGS (=3x SEQ ID NO: 92)) and anti- TCR Vα24Jα18 scFv to C-terminus of the light chain of the anti-BBGL-1/BBGL-2 Fab domain (see FIG. 5). Vα24Jα18 encodes the TCR-alpha chain of invariant NKT cells, and humanized anti- TCR Vα24Jα18 mAb was utilized to generate scFv sequences. Null Fc fragments are incorporated using modified IgG4 or IgG2 fragments.

[0314] Sequence of Valpha24 might be as follows:

[0315] TVA24_HUMAN T cell receptor alpha variable (residues 23-114)

TABLE-US-00007 ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVM TLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAF (SEQ ID  NO:81).

[0316] Sequence of TRAJ18, T cell receptor alpha joining 18:

TABLE-US-00008 DRGSTLGRLYFGRGTQLTVWP (SEQ ID NO:82).

[0317] Together: TCR Vα24Jα18

TABLE-US-00009 ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVM TLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFDRGSTLGR LYFGRGTQLTVWP (SEQ ID NO:83).

[0318] Anti- human TCR Vα24Jα18 mAb 6B11 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864538/) was humanized. The 6B11 sequence is listed below, with IMGT CDRs underlined:

[0319] VL:

TABLE-US-00010 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYW ASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGG GTKLEIK (SEQ ID NO:58)

TABLE-US-00011 6B11 VL CDR1: QDVSTA (SEQ ID NO:84), 6B11 VL CDR2: WAS (SEQ ID NO:85), 6B11 VL CDR3: QQHYSTPWT (SEQ ID NO:86).

TABLE-US-00012 6B11 VH CDR1: GFTFSNYW (SEQ ID NO:87), 6B11 VH CDR2: IRLKSNNYAT (SEQ ID NO:88), 6B11 VH CDR3: TRNGNYVDYAMDY (SEQ ID NO:89).

[0320] VH:

TABLE-US-00013 EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE IRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTR NGNYVDYAMDYWGQGTSVTVSS (SEQ ID NO:61)

[0321] Potentially immunogenic peptides were aimed to be minimized in the humanized sequences.

[0322] Humanizing mutations were made based on identity to the closest human germline sequence and predicted ability to have high protein stability, retain high affinity binding to target antigen, while minimizing potential aggregation and minimizing potential immunogenicity. Final constructs are presented in Table 1 and Table 2.

[0323] A previously humanized 6B11, mAb NKTT120, was included for comparison.

TABLE-US-00014 human TCR Vα24Jα18 VL sequences Construct Sequence % human germline Predicted immunogenic peptides Notes m6B11 VL DIVMTQSHKFMST SVGDRVSITCKASQ DVSTAVAWYQQK PGQSPKLLIYWAST RHTGVPDRFTGSG SGTDYTLTISSVQAE 69.5% IGKV1-39*01 DIVMTQSHKFMSTS V (amino acids 1-15 of SEQ ID NO:; 58) DLALYYCQQHYSTP WTFGGGTKLEIK (SEQ ID NO:58) LLIYWASTRHTGVPD (amino acids 46-60 of SEQ ID NO: 58) NKTT120 VL DIQMTQSPSSLSAS VGDRVTITCKASQD VSTAVAWYQQKP GQAPRLLIYWASTR HTGVPSRFSGSGSG TDFTLTISSLQPEDF ALYYCQQHYSTPW TFGQGTKLEIK (SEQ ID NO:59) 83.2% IGKV1-39*01 LLIYWASTRHTGVPS (amino acids 46-60 of SEQ ID NO: 59) VTCR-L1 DIQMTQSPSSLSAS VGDRVTITCRASQD VSTAVAWYQQKP GKAPKLLIYWASTR HTGVPSRFSGSGSG TDYTLTISSLQPEDF ATYYCQQHYSTPW TFGGGTKVEIK (SEQ ID NO:60) 86.3% IGKV1-39*01 LLIYWASTRHTGVPS (amino acids 46-60 of SEQ ID NO: 60)

TABLE-US-00015 Anti- human TCR Va24Ja18 VH sequences Construct Sequence % human germline Predicted immunogenic peptides Notes m6B11 VH EVKLEESGGGLVQ PGGSMKLSCVAS GFTFSNYWMNW VRQSPEKGLEWV AEIRLKSNNYATH YAESVKGRFTISRD DSKSSVYLQMNN LRAEDTGIYYCTRN GNYVDYAMDYW GQGTSVTVSS (SEQ ID NO:61) 74% IGHV3-73*01 EWVAEIRLKSNNYA T (amino acids 46-60 of SEQ ID NO: 61) DSKSSVYLQMNNLR A (amino acids 76-90 of SEQ ID NO: 61) VYLQMNNLRAEDT GI (amino acids 81-95 of SEQ ID NO: 61) NKTT120 VH EVQLVESGGGLV QPGGSLRLSCVAS GFTFSNYWMNW VRQAPGKGLEWV AEIRLKSNNYATH YAESVKGRFTISRD DSKNTVYLQMNS LRAEDTAVYYCTR NGNYVDYAMDY WGQGTLVTVSS (SEQ ID NO:62) 83% IGHV3-73*01 EWVAEIRLKSNNYA T (amino acids 46-60 of SEQ ID NO: 62) DSKNTVYLQMNSLR A (amino acids 76-90 of SEQ ID NO: 62) VYLQMNSLRAEDTA V (amino acids 81-95 of SEQ ID NO: 62) VTCR-H1 EVQLVESGGGLV QPGGSLKLSCAAS GFTFSNYWMNW VRQAPGKGLEWV AEIRLKSNNYATH YAESVKGRFTISRD DSKNTAYLQMNS LKTEDTAVYYCTR NGNYVDYAMDY WGQGTLVTVSS (SEQ ID NO:63) 88% IGHV3-73*01 EWVAEIRLKSNNYA T (amino acids 46-60 of SEQ ID NO: 63) DSKNTAYLQMNSLK T (amino acids 76-90 of SEQ ID NO: 63)

[0324] Single chain variable fragments (scFv) were designed based on a VH-VL orientation and are presented in Table 9. Additional disulfide stabilization between the VH and VL domains was engineered by substituting Cys at positions VH44-VL100 (Kabat numbering).

TABLE-US-00016 Anti- human TCR Va24Ja18 scFv sequences Construct Sequence VTCR-Chimeric-scFv EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRNGNYVDYAMDYWGQGTSVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFT GSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGGGTKLEIK (SEQ ID NO:64) VTCR-Chimeric-scFv-DS EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKCLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRNGNYVDYAMDYWGQGTSVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFT GSGSGTDYTLTISSVQAEDLALYYCQQHYSTPWTFGCGTKLEIK (SEQ ID NO:65) VTCR-NKTT120-scFv EVQLVESGGGLVQPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKNTVYLQMNSLRAEDTAVYYCTRNGNYVDYAMDYWGQGTLVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSG SGSGTDFTLTISSLQPEDFALYYCQQHYSTPWTFGQGTKLEIK (SEQ ID NO:66) VTCR-NKTT120-scFv-DS EVQLVESGGGLVQPGGSLRLSCVASGFTFSNYWMNWVRQAPGKCLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKNTVYLQMNSLRAEDTAVYYCTRNGNYVDYAMDYWGQGTLVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSG SGSGTDFTLTISSLQPEDFALYYCQQHYSTPWTFGCGTKLEIK (SEQ ID NO:67) VTCR-H1L1-scFv EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQAPGKGLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNGNYVDYAMDYWGQGTLVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSG SGSGTDYTLTISSLQPEDFATYYCQQHYSTPWTFGGGTKVEIK (SEQ ID NO:68) VTCR-H1L1-scFv-DS EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQAPGKCLEWVAEIRLKSNNYATHY AESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNGNYVDYAMDYWGQGTLVTVSS-GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS-DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSG SGSGTDYTLTISSLQPEDFATYYCQQHYSTPWTFGCGTKVEIK (SEQ ID NO:69)

TABLE-US-00017 Sequences of anti-BBGL1 and anti-BBGL-2 constructs with NKT cell effector function. CLK = kappa CL domain, CLL = lambda CL domain Construct Sequence of Chain 1 Sequence of Chain 2 Sequence of Chain 3 Sequence of Chain 4 VIT-206 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92) -(anti-Anti-Vα24Jα18 TCR scFv) Same as Chain 1 (BBGL1-VH)-(V-IGG4-A or V-IGG2 or V-IGG2-C) Same as Chain 3 VIT-207 (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(anti-Anti-Vα24Jα18 TCR scFv) Same as Chain 1 (BBGL2-VH)-(V-IGG4-A or V-IGG2 or V-IGG2-C) Same as Chain 3 VIT-208 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(anti-Anti-Vα24Jα18 TCR scFv) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(anti-Anti-Vα24Jα18 TCR scFv) (BBGL1-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (BBGL2-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT-209 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(anti-Anti-Vα24Jα18 TCR scFv) (BBGL1-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (BBGL2-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT210 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL)-GGGGSGGGGSGG GGSGGGGS(=4X SEQ ID NO: 92)-(anti-Anti-Vα24Jα18 TCR scFv) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) (BBGL1-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (BBGL2-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E)

Example 5: Design of Anti-BBGL-1 and Anti-BBGL-2 Antibodies with T Cell, NK Cell, and NKT Cell Engager Effector Functions

[0325] To more specifically activate NKT cells and to a lesser degree T cells and NK cells, novel anti CD3 x CD16/CD56 x BBGL-1/BBGL2 multi-specific antibodies were designed (see FIG. 6). The anti-CD3 and anti-CD16/CD56 moieties are encoded by Fab domains, and the anti-BBGL-1/BBGL-2 moieties are encoded by scFv fragments fused by a GlySer linker (GGGGSGGGGSGGGGS (=3x SEQ ID NO: 92)) at the C terminus of the Light chain. Null Fc fragments are incorporated using modified IgG4 or IgG2 fragments.

TABLE-US-00018 Sequences of anti-BBGL1 and anti-BBGL-2 constructs with T cell, NK cell and NKT cell effector function Construct Sequence of Chain 1 Sequence of Chain 2 Sequence of Chain 3 Sequence of Chain 4 VIT-216 (anti-CD3 VLK-CLK)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92)-(BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) (anti-CD16 or CD56 VLK or VLL)-(CLK or CLL)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92)-(BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) (anti-CD3-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (anti-CD16 or CD56-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT-217 (anti-CD3 VLK-CLK)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92) -(BBGL2-VH-VLK-scFv or BBGL2-VH-VLL-scFv) (anti-CD16 or CD56 VLK or VLL)-(CLK or CLL)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92)-(BBGL2-VH-VLK-scFv or BBGL2-VH-VLL-scFv) (anti-CD3-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (anti-CD16 or CD56-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) VIT-218 (anti-CD3 VLK-CLK)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92) -(BBGL1-VH-VLK-scFv or (anti-CD16 or CD56 VLK or VLL)-(CLK or CLL)-GGGGSGGGGSGG GGS(=3x SEQ ID NO: 92)-(BBGL2- (anti-CD3-VH)-(V-IGG4-A or V-IGG2-A or V-IGG2-D) (anti-CD16 or CD56-VH)-(V-IGG4-B or V-IGG2-B or V-IGG2-E) BBGL1-VH-VLL-scFv) VH-VLK-scFv or BBGL2-VH-VLL-scFv)

Example 6: Design of Anti-BBGL1 and anti-BBGL2 Constructs With Drug Payload Delivery

[0326] 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-00019 IgG4 sequences with engineered free cys for site specific payload conjugation Construct Sequence Notes V-IGG4-ADC-A ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:70) S228P FALA (F234A, L235A) K322A Naturally contains F405 and R409 A339C V-IGG4-ADC-A-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK (SEQ ID NO:71) Same as above, but with linker and fc only V-IGG4-ADC-B ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP S228P FALA (F234A, L235A) EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCA VSNKGLPSSIEKTISKCKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:72) K322A F405L, R409K A339C V-IGG4-ADC-B-Fc GGGGSGGGGSGGGGSKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCAVSNKGLPSSIEKTISK CKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFL LYSKLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK (SEQ ID NO:73) Same as above, but with linker and fc only

[0327] Anti-BBGL1 and anti-BBGL-2 constructs with payload conjugation sites were designed and are shown in Table 13.

TABLE-US-00020 Sequences of anti-BBGL1 and anti-BBGL2 constructs with free cys for payload conjugation. CLK = kappa CL domain, CLL = lambda CL domain Construct Sequence of Chain 1 Sequence of Chain 2 Sequence of Chain 3 Sequence of Chain 4 VIT-203 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) Same as Chain 1 (BBGL1-VH) - (V-IGG4-ADC-A) Same as Chain 3 VIT-204 (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) Same as Chain 1 (BBGL2-VH) - (V-IGG4-ADC-A) Same as Chain 3 VIT-205 (BBGL1-VLL or BBGL1-VLK) - (CLK or CLL) (BBGL2-VLL or BBGL2-VLK) - (CLK or CLL) (BBGL1-VH) - (V-IGG4-ADC-A) (BBGL2-VH) -(V-IGG4-ADC-B) VIT-203sc (BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) - (V-IGG4-ADC-A-Fc) Same as Chain 1 VIT-204sc (BBGL2-VH-VLK-scFv or BBGL2-VH- Same as Chain 1 VLL-scFv)- (V-IGG4-ADC-A-Fc) VIT-205sc (BBGL1-VH-VLK-scFv or BBGL1-VH-VLL-scFv) - (V-IGG4-ADC-A-Fc) (BBGL2-VH-VLK-scFv or BBGL2-VH-VLL-scFv)- (V-IGG4-ADC-B-Fc)

Example 7. Fusion Proteins With IgM Constant Regions

[0328] 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.

[0329] Sequence of human IgM constant region, numbered residues 1-453, where positions 131, 135, 354 and 385 are underlined by uniprot (https://www_uniprot.org/uniprot/P01871):

TABLE-US-00021 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDI SSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKN VPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLR EGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLT TYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGER FTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATIT CLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTV SEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAG TCY (SEQ ID NO:90)

[0330] Sequence of IgM CH2-CH3-CH4 which can used for fusing to antibody fragments (Fab, scFv, VHH, etc) or targeting proteins for adding IgM effector functions (residues 105-453):

[0331] V-IGM

TABLE-US-00022 VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQ VGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGL TFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDS VTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCT VTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVT GFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEE WNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY  (SEQID NO:91)

[0332] 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-00023 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

Example 8. Synthesis of BbGL1, BbGL2, BbGL1-Biotin, and BbGL2-Biotin

BbGL1

[0333] See FIG. 8

1,2,3,4,6-penta-O-benzoyl-D-galactose (2)

[0334] D-galactose (1.50 g, 8.32 mmol) was suspended in dry pyridine (25 mL). Benzoyl chloride (7.7 mL, 66 mmol) was added dropwise at 0° C. and the mixture was stirred overnight while warming to room temperature. The solvent was evaporated and the mixture was redissolved in dichloromethane (50 mL), washed with sat. NaHCO.sub.3 (2×20 mL) and sat. NaCl (2×20 mL) and dried over MgSO.sub.4. The solvent was evaporated and the product was purified by flash chromatography on silica gel using a gradient of 0-30% ethyl acetate in hexanes as the eluent. Yield: 4.47 g (77%).

[0335] .sup.1H-NMR (300 MHz, CDCl.sub.3): δ (ppm) 8.11 (m, 4H), 7.96 (m, 2H), 7.84 (m, 4H), 7.65 (m, 2H), 7.57-7.23 (m, 13H), 6.95 (d, J = 3.6 Hz, 1H), 6.19 (dd, J = 0.9 Hz, 3.4 Hz, 1H), 6.11 (d, J = 3.2 Hz, 1H), 6.04 (d, J=3.5 Hz, 1H) 4.83 (t, J=6.7 Hz, 1H), 4.63 (dd, J=6.4 Hz, 11.4 Hz, 1H), 4.42 (dd, J=7.0 Hz, 11.2 Hz, 1H).

[0336] LC-MS (ESI): Calculated for C.sub.41H.sub.32O.sub.11: 700.19; Found: 723.35 (M + Na.sup.+). Purity: > 99%

2,3,4,6-tetra-O-benzoyl-D-galactose (3)

[0337] 1,2,3,4,6-penta-O-benzoyl-D-galactose (2, 4.47 g, 6.38 mmol) was dissolved in dry dichloromethane (25 mL). 33% HBr in acetic acid (12.5 mL, 51 mmol) was added dropwise at 0° C. and the mixture was stirred overnight while warming to room temperature. The mixture was diluted with dichloromethane (50 mL), washed with sat. NaHCO.sub.3 (3×50 mL) and sat. NaCl (2×25 mL) and dried over MgSO.sub.4. The solvent was evaporated and the resulting white solid was dissolved in a mixture of acetone (20 mL) and water (2 mL). Silver carbonate (2.29 g, 8.29 mmol) was added and the mixture was stirred at room temperature for 30 min. Solids were filtered off over celite and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a gradient of 0-100% ethyl acetate in hexanes as the eluent. Yield: 3.27 g (86%).

[0338] .sup.1H-NMR (300 MHz, CDCl.sub.3): δ (ppm) 8.12-7.95 (m, 6H), 7.79 (m, 10H), 7.28-7.21 (m, 2H), 6.07 (m, 2H), 5.85 (t, J = 3.6 Hz, 1H), 5.77-5.67 (m, 1H) 4.88 (t, J=6.5 Hz, 1H), 4.62 (dd, J=6.6 Hz, 11.4 Hz, 1H), 4.40 (dd, J=6.5 Hz, 11.2 Hz, 1H), 2.97 (dd, J=1.2 Hz, 3.8 Hz, 1H).

[0339] LC-MS (ESI): Calculated for C.sub.34H.sub.28O.sub.10: 596.17; Found: 619.23 (M + Na.sup.+). Purity: 98%.

2,3,4,6-tetra-O-benzoyl-D-galactopyranosyl Trichloroacetimidate (4)

[0340] 2,3,4,6-tetra-O-benzoyl-D-galactose (3, 3.27 g, 5.48 mmol) was dissolved in dry dichloromethane (15 mL). The mixture was cooled to 0° C. and trichloroacetonitrile (2.75 mL, 27 mmol) was added, followed by 1,8-Diazabicyclo[5.4.0]undec-7-ene (0.41 mL, 2.74 mmol). The mixture was stirred at 0° C. for 30 min, concentrated by rotary evaporation, and purified by flash chromatography on silica gel using a gradient of 0-30% ethyl acetate in hexanes as the eluent. Yield: 3.36 g (83%).

[0341] .sup.1H-NMR (300 MHz, CDCl.sub.3): δ (ppm) 8.63 (s, 1H), 8.09 (m, 2H), 7.97 (m, 4H), 7.81 (m, 2H), 7.64 (m, 1H), 7.58-7.26 (m, 11H), 6.92 (d, J = 3.8 Hz, 1H), 6.16 (m, 1H), 6.06 (d, J = 3.1 Hz, 1H), 5.98 (d, J=3.6 Hz, 1H) 4.87 (t, J=6.6 Hz, 1H), 4.62 (dd, J=6.8 Hz, 11.4 Hz, 1H), 4.44 (dd, J=6.0 Hz, 11.4 Hz, 1H).

[0342] LC-MS (ESI): Calculated for C.sub.36H.sub.28Cl.sub.3NO.sub.lo: 739.08; Found: 762.18 (M + Na.sup.+). Purity: 91%.

Cholesteryl 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranoside (5)

[0343] 2,3,4,6-tetra-O-benzoyl-D-galactopyranosyl trichloroacetimidate (4, 3.36 g, 4.53 mmol) and cholesterol (1.93 g, 4.99 mmol) were dissolved in dry dichloromethane (45 mL). 4 Å molecular sieve (powder, 1 g) was added and the mixture was stirred for 10 min. A solution of trimethylsilyl trifluoromethanesulfonate (0.16 mL, 0.91 mmol) in dichloromethane (5 mL) was added dropwise. The mixture was stirred at room temperature for 1 h, quenched with triethylamine, and filtered over celite. The solvent was evaporated and the product was purified by flash chromatography on silica gel using a gradient of 0-30% ethyl acetate in hexanes as the eluent. Yield: 3.57 g (82%).

[0344] .sup.1H-NMR (300 MHz, CDCl.sub.3): δ (ppm) 8.10 (m, 2H), 8.03 (m, 2H), 7.96 (m, 2H), 7.79 (m, 2H), 7.65-7.35 (m, 12H), 5.97 (d, J = 3.5 Hz, 1H), 5.77 (dd, J=8.0 Hz, 10.4 Hz, 1H), 5.58 (dd, J = 3.5 Hz, 10.4 Hz, 1H), 5.22 (d, J=4.7 Hz, 1H) 4.90 (t, J=8.1 Hz, 1H), 4.67 (dd, J=6.9 Hz, 11.2 Hz, 1H), 4.42 (dd, J=6.4 Hz, 11.2 Hz, 1H), 4.31 (t, J=6.7 Hz, 1H), 3.55 (s, br, 1H), 2.18 (d, J=7.7 Hz, 2H), 2.05-0.85 (m, 38H), 0.65 (s, 3H).

Cholesteryl-β-D-galactopyranoside (6)

[0345] Cholesteryl 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranoside (5, 3.57 g, 3.70 mmol) was dissolved in a mixture of dry THF (50 mL) and dry methanol (50 mL). Sodium methoxide (200 mg, 3.70 mmol) was added and the mixture was stirred at room temperature for 2.5 h. The reaction was quenched with acetic acid (0.21 mL, 3.70 mmol) and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a gradient of 0-20% methanol in dichloromethane as the eluent. Yield: 1.84 g (91%).

[0346] .sup.1H-NMR (300 MHz, DMSO): δ (ppm) 5.32 (d, J=3.9 Hz, 1H) 4.71 (d, J=4.0 Hz, 1H), 4.64 (d, J=4.8 Hz, 1H), 4.51 (t, J=5.6 Hz, 1H), 4.29 (d, J=4.6 Hz, 1H), 4.17 (d, J=7.0 Hz, 1H), 3.60 (s, br, 1H), 2.45-0.80 (m, 39H), 0.65 (s, 3H).

[0347] LC-MS (ESI): Calculated for C.sub.33H.sub.56O.sub.6: 548.41; Found: 571.41 (M + Na.sup.+).

BbGL1

[0348] Palmitic acid (561 mg, 2.19 mmol) and TBTU (702 mg, 2.19 mmol) were dissolved in dry pyridine (30 mL). N,N-diisopropylethylamine (0.64 mL, 3.64 mmol) was added and the mixture was stirred at room temperature for 30 min. A solution of cholesteryl-β-D-galactopyranoside (6, 1.00 g, 1.82 mmol) in dry pyridine (10 mL) was added and the mixture was stirred at room temperature for 3 days. The solvent was evaporated and the product was purified by flash chromatography on silica gel using a gradient of 0-70% ethyl acetate in hexanes as the eluent. The purified product was dissolved in diethyl ether (50 mL) and washed with sat. NaHCO.sub.3 (2×20 mL) and sat. NaCl (2×20 mL). The combined aqueous phases were back-extracted with diethyl ether. The combined organic phases were dried over MgSO.sub.4 and the solvent was evaporated. Yield: 523 mg (37%).

[0349] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.35 (d, J=4.8 Hz, 1H), 4.33 (m, 3H), 3.88 (s, br, 1H), 3.62 (m, 4H), 2.82 (s, br, 1H), 2.68 (s, br, 1H), 2.55 (s, br, 1H), 2.32 (m, 4H), 2.06-0.80 (m, 70H), 0.68 (s, 3H).

[0350] MS (ESI): Calculated for C.sub.49H.sub.86O.sub.7: 786.64; Found: 809.59 (M + Na.sup.+)

BbGL1-Biotin

[0351] See FIG. 9

6-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoyl]-1-cholesteryl-β-D-galactopyranoside (7)

[0352] 12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoic acid (250 mg, 0.57 mmol) and TBTU (183 mg, 0.57 mmol) were dissolved in dry pyridine (6 mL). N,N-diisopropylethylamine (0.17 mL, 0.95 mmol) was added and the mixture was stirred at room temperature for 1 h. A solution of cholesteryl-β-D-galactopyranoside (6, 261 mg, 0.47 mmol) in dry pyridine (10 mL) was added and the mixture was stirred at room temperature for 2 days. The solvent was evaporated and the product was purified by flash chromatography on silica gel using a gradient of 0-75% ethyl acetate in hexanes as the eluent. Yield: 283 mg (61%).

[0353] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 7.76 (d, J=7.4 Hz, 2H), 7.59 (d, J=7.5 Hz, 1H), 7.40 (t, J=7.3 Hz, 1H), 7.31 (t, J=7.6 Hz, 1H), 5.33 (s, br, 1H), 4.82 (s, br, 1H),4.48-4.16 (m, 6H), 3.90 (s, br, 1H), 3.72-3.46 (m, 4H), 3.18 (q, J=6.5 Hz, 2H), 2.31 (m, 5H), 1.70-0.82 (m, 70H), 0.67 (s, 3H).

[0354] LC-MS (ESI): Calculated for C.sub.60H.sub.89NO.sub.9: 967.65; Found: 990.62 (M + Na.sup.+).

BbGL1-Biotin

[0355] Compound 7 (40 mg, 0.041 mmol) was dissolved in dry DMF (1 mL). 1,8-Diazabicyclo[5.4.0]undec-7-ene (7 .Math.L, 0.045 mmol) was added at 0° C. and the mixture was stirred at 0° C. for 25 min. Biotin-NHS was added, the mixture was warmed to room temperature, and stirred for 2 h. 5 .Math.L extra 1,8-Diazabicyclo[5.4.0]undec-7-ene was added and the mixture was stirred for another 30 min. The reaction mixture was diluted with THF and purified by chromatography on Bio-Beads S-X3 using THF as the eluent. Yield: 27 mg (68%).

[0356] .sup.1H-NMR (300 MHz DMSO): δ (ppm) 6.39 (d, J=17.8 Hz, 1H), 5.32 (s, br, 1H), 4.81 (t, J=4.4 Hz, 2H), 4.61 (d, J=4.5 Hz, 1h), 4.34-3.92 (m, 5H), 3.58 (s, br, 2H), 3.14-2.76 (m, 2H), 2.40-1.70 (m, 9H), 1.65-0.80 (m, 73H), 0.65 (s, 3H).

[0357] MS (ESI): Calculated for C.sub.55H.sub.93N.sub.3O.sub.9S: 971.66; Found: 972.73 (M + H+)

BbGL2

[0358] See FIG. 10

Allyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (9)

[0359] Allyl α-D-galactopyranoside (1.50 g, 6.81 mmol) was dissolved in dry pyridine (45 mL). Methoxyacetyl chloride (3.4 mL, 37.2 mmol) was added dropwise at 0° C. The mixture was stirred for 2 h while warming to room temperature. Ethyl acetate (150 mL) was added and the mixture was washed with water (2×150 mL), sat. CuSO.sub.4 (3×50 mL), sat. NaHCO.sub.3 (1×50 mL), and sat. NaCl (1×50 mL). The mixture was dried over MgSO.sub.4 and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a gradient of 0-75% ethyl acetate in hexanes as the eluent. Yield: 2.40 g (69%).

[0360] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.86 (m, 1H), 5.56-5.49 (m, 2H), 5.35-5.13 (m, 5H), 4.37-3.83 (m, 15H) 3.48-3.41 (m, 12H).

[0361] LC-MS (ESI): Calculated for C.sub.21H.sub.32O.sub.14: 508.47; Found: 531.26 (M + Na.sup.+).

(2′R)-2′,3′-Epoxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (10)

[0362] Allyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (9, 2.40 g, 4.72 mmol) was dissolved in dry dichloromethane (50 mL). m-chloroperoxybenzoic acid (2.04 g, 11.8 mmol) was added and the mixture was stirred at RT for 3 days. The mixture was diluted with dichloromethane (100 mL) and washed with sat. Na.sub.2S.sub.2O.sub.5 (50 mL), sat. NaHCO.sub.3 (50 mL), and water (50 mL). The organic phase was dried over MgSO.sub.4, concentrated by rotary evaporation, and purified by flash chromatography on silica gel using a gradient of 0-75% ethyl acetate in hexanes as the eluent. Yield: 1.66 g (67%).

[0363] (S,S)-(+)-N,N′ -Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II) (61 mg, 0.10 mmol) and p-toluenesulfonic acid monohydrate (21.22 g, 0.11 mmol) were dissolved in dichloromethane (1.7 mL) and stirred at room temperature for 30 min. The solvent was evaporated and the mixture was redissolved in dry THF (3 mL) and added to the racemic epoxidation product (2.66 g, 5.07 mmol). The mixture was cooled to 0° C., water (50 .Math.L, 2.79 mmol) was added and the mixture was stirred overnight while warming to room temperature. The solvent was evaporated and the product was purified by flash chromatography on silica gel using a gradient of 0-100% ethyl acetate in hexanes as the eluent. Yield: 1.34 g (50%).

[0364] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.57-5.47 (m, 2H), 5.26-5.18 (m, 2H), 4.37 (t, J=6.7 Hz, 1H), 4.28-3.84 (m, 12H) 3.49-3.40 (m, 12H), 3.18 (m, 1H), 2.82 (t, J=4.5 Hz, 1H), 2.61 (dd, J=2.6 Hz, 4.9 Hz, 1H).

[0365] LC-MS (ESI): Calculated for C.sub.21H.sub.32O.sub.15: 524.17; Found: 547.38 (M + Na.sup.+).

(2′R)-3′-Bromo-2′-hydroxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (11)

[0366] Lithium bromide (2 g) and Nickel bromide (2.5 g) were dissolved in dry THF (27.5 mL) and the mixture was stirred at room temperature for 2 days. The solution was allowed to settle and 9.6 mL of the supernatant was added to a solution of (2′R)-2′,3′-Epoxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (10, 1.34 g, 2.55 mmol) in dry THF (20 mL). The mixture was stirred for 1 h, diluted with dichloromethane (150 mL), and washed with water (3×50 mL). The organic phase was dried over MgSO.sub.4 and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a gradient of 0-100% ethyl acetate in hexanes as the eluent. Yield: 1.18 g (76%).

[0367] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.57-5.45 (m, 2H), 5.26-5.17 (m, 2H), 4.40 (t, J=6.8 Hz, 1H), 4.28-3.82 (m, 13H), 3.62 (dd, J=6.1 Hz, 10.6 Hz, 1H), 3.55-3.41 (m, 12H), 2.61 (d, J=5.7 Hz, 1H).

[0368] LC-MS (ESI): Calculated for C.sub.21H.sub.33BrO.sub.15: 604.10; Found: 627.31 (M + Na.sup.+).

(2′R)-3′-Bromo-2′-palmitoyloxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (12)

[0369] (2′R)-3′-Bromo-2′-hydroxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (11, 0.93 g, 1.54 mmol) was dissolved in dry dichloromethane (20 mL). Pyridine (2.48 mL, 30 mmol) was added and the mixture was cooled to 0° C. Palmitoyl chloride was added dropwise and the mixture was stirred at 0° C. for 1 h. The mixture was diluted with dichloromethane (30 mL) and washed with water (20 mL), sat. CuSO.sub.4 (2×20 mL), sat. NaHCO.sub.3 (20 mL), and sat. NaCl. The organic phase was dried over MgSO.sub.4 and concentrated by rotary evaporation. The product was purified by flash chromatography on silica gel using a gradient of 0-50% ethyl acetate in hexanes as the eluent. Yield: 1.02 g (78%).

[0370] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.56 (m, 1H), 5.47 (dd, J=3.3 Hz, 10.1 Hz, 1H), 5.24-5.08 (m, 3H), 4.35-3.48 (m, 18H), 3.55-3.42 (m, 12H), 2.36 (t, J=7.4 Hz, 2H), 1.64 (t, J=7.0 Hz, 2H), 1.38-1.18 (m, 26H), 0.88 (t, J=6.7 Hz, 3H).

[0371] LC-MS (ESI): Calculated for C.sub.37H.sub.63BrO.sub.16: 842.33; Found: 865.59 (M + Na.sup.+).

1-O-oleoyl-2-O-palmitoyl-sn-glyceryl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (13)

[0372] Tetra-n-butylammonium hydroxide solution (55% in water, 1.14 mL, 2.42 mmol) was added to a solution of oleic acid (717 mg, 2.54 mmol) in water (3 mL). The mixture was stirred overnight and the solvent was evaporated. The mixture was dried azeotropically with toluene and dissolved in dry DMF (15 mL). (2′R)-3′-Bromo-2′-palmitoyloxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (12, 1.02 g, 1.21 mmol) was added and the mixture was stirred at 80° C. for 1 h. Diethyl ether (75 mL) was added and the mixture was washed with water (2×25 mL) and sat. NaCl (25 mL). The organic phase was dried over MgSO.sub.4 and the solvent was evaporated. The product was used in the next step without purification.

BbGL2

[0373] Crude 1-O-oleoyl-2-O-palmitoyl-sn-glyceryl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (13, 1.26 g, 1.21 mmol) was dissolved in a mixture of chloroform (4.5 mL) and methanol (12 mL). t-butylamine (2.53 mL, 24 mmol) was added at 0° C. The ice bath was removed and the mixture was stirred at room temperature for 2 h. The solvent was evaporated and the mixture was purified by flash chromatography on silica gel using a gradient of 0-5% methanol in dichloromethane as the eluent. Yield: 736 mg (80% over 2 steps).

[0374] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 5.35 (m, 2H), 5.26 (m, 1H), 4.95 (d, J=3.6 Hz, 1H), 4.38 (dd, J=4.2 Hz, 11.9 Hz, 1H), 4.16-4.08 (m, 2H), 4.00-3.78 (m, 5H), 3.64 (dd, J=6.2 Hz, 10.9 Hz, 1H), 2.32 (m, 4H), 2.01 (m, 4H), 1.61 (m, 4H), 1.40-1.17 (m, 46H), 0.88 (t, J=6.7 Hz, 6H).

[0375] MS (ESI): Calculated for C.sub.43H.sub.80O.sub.10: 756.58; Found: 779.69 (M + Na.sup.+)

BbGL2-Biotin

[0376] See FIG. 11.

(2′R)-3′-Bromo-2′-[12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (14)

[0377] (2′R)-3′-Bromo-2′-hydroxypropyl 2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (11, 100 mg, 0.17 mmol) and 12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoic acid (108 mg, 0.25 mmol) were dissolved in dry dichloromethane (1 mL). Diisopropylcarbodiimide (39 .Math.L, 0.25 mmol) and 4-dimethylaminopyridine (30 mg, 0.25 mmol) were added and the mixture was stirred at room temperature for 1.5 h. The product was purified by flash chromatography on silica gel using a gradient of 0-5% methanol in dichloromethane as the eluent. Yield: 132 mg (78%).

[0378] .sup.1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 7.76 (d, J=7.4 Hz, 2H), 7.60 (d, J=7.6 Hz, 2H), 7.40 (t, J=7.2 Hz, 2H), 7.31 (dt, J=1.1 Hz, 7.4 Hz, 2H), 5.55 (d, J=3.3 Hz, 1H), 5.47 (dd, J=3.4 Hz, 10 Hz, 1H), 5.28-5.08 (m, 3H), 4.77 (s, br, 1H), 4.40 (d, J=6.9 Hz, 2H), 4.35-3.35 (m, 33H), 3.18 (m, 2H), 2.35 (t, J=7.4 Hz, 2H), 1.64 (m, 2H), 1.50 (m, 2H), 1.28 (m, 2H).

[0379] LC-MS (ESI): Calculated for C.sub.48H.sub.66BrNO.sub.18: 1023.35; Found: 1024.70 (M + H.sup.+).

(2′R)-3′-Bromo-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-a-D-galactopyranoside (15)

[0380] (2′R)-3′-Bromo-2′-[12-(9H-Fluoren-9-ylmethoxycarbonylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (14, 119 mg, 0.12 mmol) was dissolved in dry DMF (2 mL) and cooled to 0° C. 1,8-Diazabicyclo[5.4.0]undec-7-ene (26 .Math.L, 0.17 mmol) was added and the mixture was stirred at 0° C. for 25 min. Biotin (85 mg, 0.35 mmol) and HATU (132 mg, 0.35 mmol) were added and the mixture was stirred for 75 min at 0° C. Ethyl acetate (10 mL) was added and the mixture was washed with water (2×5 mL) and sat. NH.sub.4Cl (2×5 mL). The organic phase was dried over MgSO.sub.4 and the solvent was evaporated. The product was purified by flash chromatography on silica gel using a gradient of 0-10% methanol in dichloromethane as the eluent. Yield: 74 mg (62%).

[0381] LC-MS (ESI): Calculated for C.sub.43H.sub.70BrN.sub.3O.sub.18S: 1027.36; Found: 1028.70 (M + H.sup.+).

(2′R)-3′-oleoyl-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (16)

[0382] Tetra-n-butylammonium hydroxide solution (55% in water, 68 .Math.L, 0.14 mmol) was added to a solution of oleic acid (43 mg, 0.15 mmol) in water (0.3 mL). The mixture was stirred overnight and the solvent was evaporated. The mixture was dried azeotropically with toluene and dissolved in dry DMF (1 mL). (2′R)-3′-Bromo-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (15, 74 mg, 0.072 mmol) was added and the mixture was stirred at 80° C. for 1 h. Ethyl acetate (10 mL) was added and the mixture was washed with water (2×5 mL) and sat. NaCl (2×5 mL). The organic phase was dried over MgSO.sub.4 and the solvent was evaporated. The product was used in the next step without purification

[0383] LC-MS (ESI): Calculated for C.sub.61H.sub.103N.sub.3O.sub.20S: 1229.69; Found: 1231.05 (M + H.sup.+).

BbGL2-Biotin

[0384] Crude (2′R)-3′-oleoyl-2′-[12-biotinylamino)-dodecanoyl]-2,3,4,6-tetra-O-methoxyacetyl-α-D-galactopyranoside (16, 88 mg, 0.072 mmol) was dissolved in a mixture of chloroform (0.3 mL) and methanol (0.8 mL). t-butylamine (0.15 mL, 1.43 mmol) was added at 0° C. The ice bath was removed and the mixture was stirred at room temperature for 75 min. The solvent was evaporated and the mixture was purified by flash chromatography on silica gel using a gradient of 0-20% methanol in dichloromethane as the eluent. Yield: 39 mg (58% over 2 steps).

[0385] 1H-NMR (300 MHz CDCl.sub.3): δ (ppm) 6.31 (s, br, 1H), 6.21 (s, br, 1H), 5.71 (s, br, 1H), 5.34 (m, 2H), 5.26 (m, 1H), 4.90 (d, J=3.4 Hz, 1H), 4.51 (m, 2H), 4.35 (m, 2H), 4.20-4.09 (m, 2H), 3.95-3.75 (m, 6H), 3.64 (dd, J=5.8 Hz, 10.9 Hz, 2H), 3.36-3.09 (m, 3H), 2.90 (dd, J=4.7 Hz, 12.8 Hz, 1H), 2.75 (d, J=12.8 Hz, 1H), 2.32 (q, J=7.4 Hz, 4H), 2.22 (m, 2H), 2.01 (m, 4H), 1.92-1.18 (m, 49H), 0.88 (t, J=6.7 Hz, 3H).

[0386] MS (ESI): Calculated for C.sub.49H.sub.87N.sub.3O.sub.12S: 941.60; Found: 942.89 (M + H.sup.+)

Example 9: Preparation of BBGL-1 and BBGL-2 Liposome Vaccines

[0387] The following liposome vaccines were generated with the defined compositions: [0388] VIT-GLA: DSPC/DSPG/Cholesterol/BBGL-1 (7:2:1:1 molar ratio) [0389] VIT-GLB: DSPC/DSPG/Cholesterol/BBGL-2 (7:2:1:1 molar ratio)

[0390] The liposome vaccines were generated by hydrating and mixing the glycolipids and cholesterol in chloroform and drying overnight at 30 deg C in a rotary evaporator at 120 rpm. The dried mixtures were re-hydrated in aqueous buffer (10 mM phosphate buffer pH7.4) on rotation at 50 deg C for 45 min and then subjected to successive rounds of extrusion through 400 nm, 200 nm and 100 nm membranes to generate 100-150 nm diameter unilammelar vesicles with low polydispersity indices (<0.2). Finished extruded samples are sterile filtered with a 0.22 um syringe filter into vials using aseptic technique in a Laminar Flow Hood. Vials are capped and sealed and stored at 4° C. until further use.

[0391] For DLS analysis, samples are diluted into deionized (DI) water.

Example 10: Stability Profile of Prepared Liposomes VIT-GLA and VIT-GLB by Dynamic Light scattering

[0392] The physical stability of the prepared liposomes VIT-GLA and VIT-GLB stored at 4° C. was assessed by dynamic light scattering on a Malvern Zetasizer Nano. Measurements were taken in triplicate and are shown in the tables below.

TABLE-US-00024 Size and polydispersity of VIT-GLA Day Diameter (nm) Polydispersity Index 0 157.0 0.10 7 157.1 0.09 14 156.3 0.08 22 157.0 0.08 29 157.5 0.09 38 158.4 0.09 53 156.6 0.08 70 155.6 0.08 119 158.2 0.08

TABLE-US-00025 Size and polydispersity of VIT-GLB Day Diameter (nm) Polydispersity Index 0 160.4 0.19 4 108.2 0.04 11 135.0 0.06 19 134.2 0.07 26 134.4 0.09 35 137.7 0.07 50 136.0 0.06 67 135.7 0.07 116 151.2 0.17

[0393] Both VIT-GLA and VIT-GLB were highly stable for greater than 3 months. During the 119 days of testing, VIT-GLA had an average diameter of 155.6-158.4 nm and polydispersity index 0.08-0.10. During the 116 days of testing, VIT-GLB had an average diameter of 108.2-151.2 nm and polydispersity index 0.04-0.19.

Example 11: Immunization Profile, in Vitro Efficacy, and Safety of an Adjuvanted Lyme Disease Vaccine in Mouse Model

[0394] A pilot study in mice was conducted to determine the safety and in vitro efficacy of the BBGL-1 and BBGL-2 liposome vaccines in the absence or presence of the adjuvant QS-21 (Desert King International, San Diego, CA USA). Eighty-four (42 male, 42 female) C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME USA), at approximately 7 weeks were used in the study, which is outlined in Table 17a and 17b.

TABLE-US-00026 Study outline 1 Description Group Size Vaccine (PO) Observations Weights Adjuvant Control 1 6M 1 and 30 Pre-study, 1 through 60 Pre-study, 1-7, 15, 22, 29, 36, 43, 50, 57, 60 Adjuvant Control 1 6F Immunization A 2 6M Immunization A 2 6F Adjuvant + Immunization A 3 6M Adjuvant + Immunization A 3 6F Immunization B 4 6M Immunization B 4 6F Adjuvant + Immunization B 5 6M Adjuvant + Immunization B 5 6F Immunization A+B 6 6M Immunization A+B 6 6F Adjuvant + Immunization A+B 7 6M Adjuvant + Immunization A+B 7 6F

TABLE-US-00027 Study outline 2 Blood Collection Neutralization AB response Description Group Size Bio-plex (4x) (3x) Hematology Chemistry Necropsy Adjuvant Control 1 6M Pre-study, 7, 15, 30, 45, 60 (n = 42/collection) Pre-study and 60 (n =42/collection) 60 Adjuvant Control 1 6F Immunization A 2 6M Immunization A 2 6F Adjuvant + Immunization A 3 6M Adjuvant + Immunization A 3 6F Immunization B 4 6M Immunization B 4 6F Adjuvant + Immunization B 5 6M Adjuvant + Immunization B 5 6F Immunization A+B 6 6M Immunization A+B 6 6F Adjuvant + Immunization A+B 7 6M Adjuvant + Immunization A+B 7 6F

[0395] 7 Group of Mice (6 male and 6 female in each group) were given either 1) adjuvant alone, 2) Immunization A, 3) Adjuvant + Immunization A, 4) Immunization B, 5) Adjuvant + Immunization B, 6) Immunization A + Immunization B, or 7) Adjuvant + Immunization A + Immunization B

[0396] Immunization A: DSPC/DSPG/Cholesterol/BBGL-1 (7:2:1:1) (=VIT-GLA, example 9),

[0397] Immunization B: DSPC/DSPG/Cholesterol/BBGL-2 (7:2:1:1) (=VIT-GLB, example 9).

[0398] Immunization A and Immunization B were administered at 77 .Math.g/mouse doses at day 1 and Day 30. Groups receiving adjuvant received 10 .Math.g QS-21/mouse at Day 1 and Day 30.

[0399] The following endpoints were measured during the study:

1 Observations and Body Weights

[0400] Animals were monitored for clinical signs throughout the study. Body weights were measured at baseline, day 1, 7,15,22,29,36,43,50,57 and 60.

2 Clinical Chemistry and Hematology

[0401] Blood was collected at baseline and day 60 for clinical chemistry and hematology analysis. For clinical chemistries, whole blood was stored in lithium heparin tubes until analysis.

3 Serum Collection

[0402] Whole blood was collected into serum separator tubes at baseline and days 7, 15, 30, 45, and 60. Blood was allowed to clot and centrifuged at 1500xg and 10° C. for 15 minutes. Serum was aliquoted and stored at -30° C.

5 Neutralization Assay

[0403] Serum aliquots were evaluated after 30 days, for neutralizing antibodies against Borrelia burgdorferi, B. mayonii, B. afzelii, and B. garinii using laboratory developed assays.

7 Necropsy

[0404] At day 60, animals were euthanized, and necropsy was conducted. Brain, lung, heart, liver, kidney, spleen, pancreas, esophagus, stomach, intestine, bone, ovaries, prostate, testes, spinal cord, bladder, lymph nodes were collected for H&E. Samples were stored in 10% Neutral Buffered Formalin.

[0405] In vitro neutralization of Borrelia burgdorferi from Day 30 assessed with half of the mouse serum samples (n=6 per group) are shown in FIG. 7a. At day 30, Group 1 sera (QS-21 adjuvant alone) showed >15% neutralization activity, whereas Group 7 sera (QS-21 Adjuvant + Immunization A + Immunization B) showed the highest level of neutralization activity (>30%). The average weight of the mice per group (n=12 per group) over the course of the 60 day experiment is shown in FIG. 7b. All groups showed a healthy trend of weight gain, indicating safe delivery of the vaccine test articles.

Example 12: Immunization Profile, In Vitro Efficacy, and Safety of an Adjuvanted Lyme Disease Vaccine in Mouse Model.

[0406] Following the mouse trial conducted as described in Example 11, following analysis will be performed:

2 Clinical Chemistry and Hematology

[0407] Chemistry analysis of whole blood or plasma will be performed on a Zoetis Vetscan VS2 analyzer with Comprehensive Diagnostic rotors. For hematology, whole blood was stored in EDTA tubes until analysis. Hematology analysis of whole blood will be performed on a Zoetis Vetscan HMS analyzer.

4 Immune Titers

[0408] Serum aliquots for Immune titer analysis will be tested for IgM, IgG, and IgA.

5 Neutralization Assay

[0409] Serum aliquots after 60 days will be evaluated for neutralizing antibodies against Borrelia burgdorferi,B. mayonii, B. afzelii, and B. garinii using laboratory developed assays.

6 Cytokine Analysis

[0410] Cytokine analysis will be performed on serum aliquots using the Bio-Rad Bio-Plex 200 system and Bio-Plex Pro Mouse Cytokine 23-plex Assay kits.

7 Necropsy

[0411] Brain, lung, heart, liver, kidney, spleen, pancreas, esophagus, stomach, intestine, bone, ovaries, prostate, testes, spinal cord, bladder, lymph nodes will be subjected to H&E.

Example 13: Use of Biotin-BBGL1 and Biotin-BBGL2 for Measuring Antibody Titers in Lyme Disease Patient Sera

[0412] A Meso Scale Discovery Electrochemiluminescence (MSD-ECL) assay was developed using Biotin-BBGL-1 and Biotin-BBGL-2 to measure antibody titers in Lyme Disease patient samples. The assay involved first blocking MSD streptavidin gold plate with 4% BGG (bovine gamma globulin in PBS), washing the plates with PBS and the coating Biotin-BBGL-1 and Biotin-BBGL-2 at concentration of 4 ug/mL (diluted in 4% BGG) for coating for IgG side of plate and at concentration of 1 ug/mL (diluted in 4% BGG) for coating for IgM side of plate. Serum samples (diluted 1:5) were then added to each well, incubated, and washed.

[0413] Detection reagents (Sulfo tag anti-lgG Fc or anti-IgM) were then added, incubated, and washed, followed by reading the plates on a Sector Imager (Meso Scale Diagnostic, Rockville, MD USA).

[0414] Data from 3 Lyme Disease patients is presented in Table 18 and 19 and shows that this method can detect the presence of anti-BBGL1 and anti-BBGL2 IgG and IgM titers in clinical samples.

TABLE-US-00028 Patients # Gender Etnicity Age ELISA/ Western Duration Disease Symptoms L1 M Caucasian 49 NA > 12 months Joint pain, memory loss L2 M Caucasian 43 IgG+/ IgM+ NA Arthritis L3 M Caucasian/ Indian 36 IgG+/ IgM+ 12 months Arthritis, leg/ foot pain

TABLE-US-00029 Anti-BBGL1, antiBBGL2 and IgM titers EU Fold/Baseline # BBGL1 IgM* BBGL1 IgG** BBGL2 IgM* BBGL2 IgG** BBGL1 IgM* BBGL1 IgG** BBGL2 IgM* BBGL2 IgG** L1 4055.50 218.50 3458.00 2720.00 21 1.3 18 16 L2 390.00 143.50 344.50 619.50 2 0.9 2 4 L3 1338.00 166.00 1229.50 178.00 7 1.0 6 1.1 *NC= 189.75 **NC= 166.75