Antibodies and methods for producing antibodies

Abstract

This disclosure relates, in general, to antibodies and methods for producing antibodies. Specifically, this disclosure relates to antibodies and antigen-binding fragments thereof that have modifications that help increase VH1, VH2, VL1 and VL2, as well as desired pairings of VH and VL, such as VH1 / VL1 and VH2 / VL2 rather than VH1 / VL2 and VH2 / VL1.

Description

Technical Field

[0001] (Incorporation by reference to priority application) This application claims priority to U.S. Provisional Patent Application No. 63 / 506,301, filed Jun. 5, 2023, which is hereby incorporated by reference in its entirety.

[0002] (Cross-reference to related applications) This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named PROGX.001 WOSEQLISTEQIST.XML, created on May 24, 2024 and last modified, and is 147,428 bytes in size. The information of the electronic sequence listing is hereby incorporated by reference in its entirety.

[0003] (Field of the Invention) The present disclosure generally relates to antibodies and methods of producing antibodies.

Background Art

[0004] Bispecific antibodies have been shown to be a promising strategy to overcome some of the fundamental limitations of monospecific antibody therapies that cannot address multiple cross-talking signaling pathways underlying a target disease. Efforts have been made to address the immunogenicity associated with multivalent molecules, the problem of chain mispairing, and the scalability of manufacturing, but there remains a need to develop bispecific formats that effectively address these issues. Brinkmann U, Kontermann RE. 2017. 「The making of bispecific antibodies.」 mAbs 9:182-212; Godar et Godar M, de Haard H, Blanchetot C, Rasser J. 2018. 「Therapeutic bispecific antibody formats: a patent applications review」 (1994-2017). Expert. Opin. Ther. Pat. 28:251-276.

Summary of the Invention

[0005] Some embodiments provided herein are described by the following numbered embodiments, and are also provided as possible combinations or overlapping embodiments. 1. An antigen-binding fragment (Fab), The human IgG heavy chain (HC) variable domain (VH), the human IgG heavy chain (HC) constant domain (CH1), the human IgG light chain (LC) variable domain (VL), and the human IgG light chain (LC) constant domain (CL) are included, wherein HC CH1 contains substitutions in F170, S183, and V185 (EU numbering), LC CL contains substitution in L135 (EU numbering), and HC1 does not contain any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (European numbering). 2. The Fab according to Embodiment 1, wherein CH1 consists of substitutions in F170, S183, and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). 3. The Fab according to any one of Embodiments 1 to 2, wherein the HC CH1 substitution in F170 (EU numbering) is F170I or F170V substitution. 4. The Fab according to any one of Embodiments 1 to 3, wherein the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. 5. Fab as described in any one of Embodiments 1 to 4, where HC CH1 substitution in V185 (EU numbering) is V185L substitution. 6. Fab according to any one of Embodiments 1 to 5, in which the LC CL1 substitution in L135 (EU numbering) is L135F substitution. 7. The Fab according to Embodiment 1 or 2, wherein HC CH1 includes or consists of F170V, S183I, and V185L (EU numbering) substitutions, and LC CL includes or consists of L135F (EU numbering) substitution. 8. The Fab according to Embodiment 1 or 2, wherein HC CH1 includes or consists of the substitutions F170I, S183L, and V185L (EU numbering), and LC CL includes or consists of the substitution L135F (EU numbering). 9. Antigen-binding fragment (Fab), The human IgG heavy chain (HC) variable domain (VH), human IgG heavy chain (HC) constant domain (CH1), human IgG light chain (LC) variable domain (VL), and human IgG light chain (LC) constant domain (CL), wherein HC CH1 includes substitutions of F126, F170, S183, V185, and C220 (EU numbering), LC CL includes substitutions of E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and optionally, LC CL includes substitutions of E124, L135, and C214 (lambda), or optionally, LC CL includes substitutions for Q124, L135, and C214 (Kappa), while HC1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 10. The Fab according to Embodiment 9, wherein CH1 consists of substitutions in F126, F170, S183, V185, and C220 (EU numbering), and LC CL includes substitutions in E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), optionally including substitutions in E124, L135, and C214 (lambda), or optionally including substitutions in Q124, L135, and C214 (kappa). 11. The Fab according to Embodiment 9 or 10, in which the HC CH1 substitution in F126 (EU numbering) is F126C substitution. 12. The Fab according to any one of Embodiments 9 to 11, wherein the HC CH1 substitution in F170 (EU numbering) is F170V or F170I substitution. 13. The Fab according to any one of Embodiments 9 to 12, wherein the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. 14. Fab as described in any one of Embodiments 9 to 13, where HC CH1 substitution in V185 (EU numbering) is V185L substitution. 15. Fab as described in any one of embodiments 9 to 14, where HC CH1 substitution in C220 (EU numbering) is C220S substitution. 16. The Fab according to any one of Embodiments 9 to 15, wherein the LC CL substitution in E124 (lambda) or Q124 (kappa) (EU numbering) is E124C or Q124C substitution, and optionally, the LC CL substitution in E124 (lambda) is E124C substitution, or optionally, the LC CL substitution in Q124 (kappa) is Q124C substitution. 17. LC CL substitution in L135 (EU numbering) is L135F substitution, as described in any one of embodiments 9 to 16 of Fab. 18. LC CL substitution in C214 (EU numbering) is C214S substitution, as described in any one of embodiments 9 to 17 of Fab. 19. The Fab according to Embodiment 9, wherein HC CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering), optionally including substitutions for E124C, L135F, and C214S (lambda) (EU numbering), or optionally including substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). 20. The Fab according to Embodiment 9, wherein HC CH1 includes or consists of substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes substitutions E124C, L135F, and C214S (lambda) or Q124, L135, and C214 (kappa) (EU numbering), optionally including substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally including substitutions Q124, L135, and C214 (kappa) (EU numbering). 21. Antigen-binding fragment (Fab), It comprises a human IgG heavy chain (HC) variable domain (VH), a human IgG heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a human IgG light chain (LC) constant domain (CL), wherein HC CH1 includes substitutions of F126 and C220 (EU numbering), LC CL includes substitutions of E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering), optionally, LC CL includes substitutions of E124 and C214 (lambda) (EU numbering), or optionally, LC CL includes substitutions of Q124 and C214 (kappa) (EU numbering). HC1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170I, F170M, F170Y, F170S, F170A, F170V, S181I, S181T, S181M, S183A, S183L, S183I, S183V, V185L and V185A (EU numbering), CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (European numbering). 22. The Fab according to Embodiment 21, wherein the HC CH1 substitution at F126 (EU numbering) is F126C substitution, the LC CL substitution at position 124 (EU numbering) is E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC CL substitution at position 124 (EU numbering) is E124C (lambda) substitution, or optionally, the LC CL substitution at position 124 (EU numbering) is Q124C (kappa) substitution. 23. The Fab according to any one of embodiments 21 to 22, wherein the HC CH1 substitution in C220 (EU numbering) is C220S substitution. 24. LC CL substitution in C214 (EU numbering) is C214S substitution, as described in any one of embodiments 21 to 23. 25. The Fab according to any one of Embodiments 1 to 24, wherein HC CH1 includes or consists of SEQ ID NO: 15 or 19, LC CL includes or consists of SEQ ID NO: 2 or 7, or HC CH1 includes or consists of SEQ ID NO: 23 or 24, and LC CL includes or consists of SEQ ID NO: 5 or 8. 26. An antibody comprising the Fab described in any one of Embodiments 1 to 25. 27. The antibody according to Embodiment 26, further comprising a second Fab, wherein one of the Fabs, but not both, comprises HC CH1 with substitutions at F126 and C220 (EU numbering), and LC CL with substitutions at E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering), optionally, LC CL comprising substitutions at E124 and C214 (lambda) (EU numbering), or optionally, LC CL comprising substitutions at Q124 and C214 (kappa) (EU numbering). 28. The antibody according to Embodiment 27, wherein the HC CH1 substitution at F126 (EU numbering) is an F126C substitution, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC CL substitution at position 124 (EU numbering) is a Q124C (kappa) substitution. 29. The antibody according to any one of Embodiments 27 to 28, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution. 30. The antibody according to any one of Embodiments 27 to 29, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution. 31. An antibody comprising one or more IgG Fabs having wild-type CH and wild-type CL, and one or more Fabs described in any one of Embodiments 1 to 30. 32. The antibody according to any one of Embodiments 27 to 31, wherein the antibody comprises the Fab described in any one of Embodiments 1 to 8 and the Fab described in any one of Embodiments 21 to 24, or the Fab described in any one of Embodiments 9 to 20, and the Fab that does not contain HC CH1 with substitutions at F126 and C220 (EU numbering), and the Fab that does not contain LC CL with substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), and optionally, the LC CL includes substitutions at position E124 and C214 (EU numbering) for lambda, or optionally, the LC CL includes substitutions at position Q124 and C214 (EU numbering) for kappa. 33. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), The first and second (LC2) light chains are each comprising the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1), and the second and second (LC2) light chains are each comprising the human IgG light chain variable domain (VL) and the light chain constant domain (CL). HC1 CH1 includes substitutions for F170, S183, and V185 (EU numbering), LC1 CL includes substitutions for L135 (EU numbering), and HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). LC1 CL is an antibody or its fragment that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 34. The antibody or fragment thereof according to Embodiment 33, wherein HC1 CH1 consists of substitutions in F170, S183, and V185 (EU numbering), and LC1 CL consists of substitutions in L135 (EU numbering). 35. The antibody or fragment according to Embodiment 33, wherein HC2 CH1 contains or comprises SEQ ID NO: 9, and LC2 CL contains or comprises SEQ ID NO: 1 or 6. 36. The antibody or fragment thereof according to Embodiment 33, wherein HC1 CH1 is substituted with V185. 37. An antibody or fragment thereof according to any one of embodiments 33 to 35, wherein the HC1 CH1 substitution in F170 (EU numbering) is an F170I or F170V substitution. 38. An antibody or fragment thereof according to any one of embodiments 33 to 36, wherein the HC1 CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. 39. An antibody or fragment thereof according to any one of Embodiments 33 to 37, wherein the HC1 CH1 substitution in V185 (EU numbering) is a V185L substitution. 40. An antibody or fragment thereof according to any one of embodiments 33 to 38, wherein the LC1 CL1 substitution in L135 (EU numbering) is an L135F substitution. 41. The antibody or fragment thereof according to Embodiment 33, wherein HC1 CH1 contains or consists of F170V, S183I and V185L (EU numbering) substitutions, and LC1 CL contains or consists of L135F (EU numbering) substitution. 42. The antibody or fragment thereof according to Embodiment 33, wherein HC1 CH1 contains or consists of F170I, S183L, and V185L (EU numbering) substitutions, and LC1 CL contains or consists of L135F (EU numbering) substitution. 43. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), The first and second (LC2) light chains are each comprising the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1), and the second and second (LC2) light chains are each comprising the human IgG light chain variable domain (VL) and the light chain constant domain (CL). HC1 CH1 includes or consists of substitutions for F126, F176, S189, V191, and C220 (EU numbering), and LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering). An antibody or fragment thereof in which HC1 CH1 does not contain any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and LC1 CL does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 44. The antibody or fragment thereof according to Embodiment 43, wherein the HC CH1 substitution in F126 (EU numbering) is an F126C substitution. 45. The antibody or fragment thereof according to any one of embodiments 43 to 44, wherein the HC CH1 substitution at F170 (EU numbering) is an F170V or F170I substitution. 46. The antibody or fragment thereof according to any one of embodiments 43 to 45, wherein the HC CH1 substitution at S183 (EU numbering) is an S183L or S183I substitution. 47. The antibody or fragment thereof according to any one of embodiments 43 to 46, wherein the HC CH1 substitution at V185 (EU numbering) is a V185L substitution. 48. The antibody or fragment thereof according to any one of embodiments 43 to 47, wherein the HC CH1 substitution at C220 (EU numbering) is a C220S substitution. 49. The antibody or fragment thereof according to any one of embodiments 43 to 48, wherein the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, optionally, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC CL substitution at position 124 (EU numbering) is a Q124C (kappa) substitution. 50. The antibody or fragment thereof according to any one of embodiments 43 to 49, wherein the LC CL substitution at L135 (EU numbering) is an L135F substitution. 51. The antibody or fragment thereof according to any one of embodiments 43 to 50, wherein the LC CL substitution at C214 (EU numbering) is a C214S substitution. 52. The antibody or fragment thereof according to Embodiment 43, wherein HC CH1 contains or comprises the substitutions F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL contains or comprises the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally comprising or comprising the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally comprising or comprising the substitutions Q124C, L135F, and C214S (kappa) (EU numbering). 53. The antibody or fragment thereof according to Embodiment 43, wherein HC CH1 contains or comprises the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL contains or comprises the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally comprising the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally comprising the substitutions Q124C, L135F, and C214S (kappa) (EU numbering). 54. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), The first and second (LC2) light chains are each comprising the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1), and the second and second (LC2) light chains are each comprising the human IgG light chain variable domain (VL) and the light chain constant domain (CL). HC1 CH1 contains or consists of substitutions at F126 and C220 (EU numbering), LC1 CL contains or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 contains or consists of substitutions at F170, S183, and V185 (EU numbering), LC2 CL contains or consists of substitution L135 (EU numbering), and HC1 CH1 and / or HC2 CH1 do not contain any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). An antibody or fragment thereof, wherein LC1 CL and / or LC2 CL do not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 55. The antibody according to embodiment 54, wherein the HC1 CH1 substitution at F126 (EU numbering) is the F126C substitution, the LC1 CL substitution at position 124 (EU numbering) is the E124C substitution for lambda or the Q124C substitution for kappa LC, and optionally, the LC1 CL substitution at position 124 (EU numbering) is the E124C substitution for lambda or optionally, the LC1 CL substitution at position 124 (EU numbering) is the Q124C substitution for kappa LC. 56. The antibody according to any one of embodiments 54 - 55, wherein the HC CH1 substitution at C220 (EU numbering) is the C220S substitution. 57. The antibody according to any one of embodiments 54 - 56, wherein the LC CL substitution at C214 (EU numbering) is the C214S substitution. 58. An antibody or fragment thereof according to any one of Embodiments 26 to 57, wherein the antibody comprises two heavy / light chain pairs, the first pair being HC1 / LC1 and the second pair being HC2 / LC2. 59. An antibody according to any one of embodiments 26 to 57, further comprising a pair of HC1 and HC2. 60. The antibody according to Embodiment 59, wherein HC1 and HC2 are paired via charge-charge interactions. 61. The antibody according to any one of embodiments 26 to 60, wherein each HC1 and HC2 further comprises a heavy chain constant domain (CH3), and the CH3 domain of HC1 and the CH3 domain of HC2 together constitute a knob-into-hole construct. 62. The antibody according to embodiment 61, wherein HC1 CH3 constitutes a knob and HC2 CH3 constitutes a hole. 63. The antibody according to embodiment 61, wherein HC1 CH3 constitutes a hole and HC2 CH3 constitutes a knob. 64. The antibody according to any one of embodiments 62 to 63, wherein HC1 CH3 and / or HC2 CH3 include or consist of T366, L368, and Y407 (EU numbering) substitutions. 65. The antibody according to Embodiment 64, wherein the HC1 CH3 and / or HC2 CH3 substitution in T366 (EU numbering) is a T366W or T366S substitution. 66. The antibody according to any one of embodiments 64 to 65, wherein the HC1 CH3 and / or HC2 CH3 substitution in L368 (EU numbering) is an L368A substitution. 67. The antibody according to any one of embodiments 64 to 66, wherein the HC1 CH3 and / or HC2 CH3 substitution in Y407 (EU numbering) is a Y407V substitution. 68. The antibody according to any one of embodiments 64 to 67, wherein HC1 CH3 contains or consists of the T366W (EU numbering) substitution, and HC2 CH3 contains or consists of the T366S, L368A, and Y407V (EU numbering) substitutions. 69. The antibody according to any one of embodiments 64 to 68, wherein HC1 CH3 contains or consists of T366S, L368A, and Y407V (EU numbering) substitutions, and HC2 CH3 contains or consists of T366W (EU numbering) substitution. 70. An antibody according to any one of embodiments 26 to 69, wherein LC1 and / or LC2 are kappa light chains. 71. An antibody according to any one of embodiments 26 to 69, wherein LC1 and / or LC2 are lambda light chains. 72. An antibody according to any one of embodiments 26 to 69, wherein LC1 is a kappa light chain and LC2 is a lambda light chain. 73. An antibody according to any one of embodiments 26 to 69, wherein LC1 is a lambda light chain and LC2 is a kappa light chain. 74. An antibody or fragment thereof according to any one of embodiments 26 to 73, wherein HC2 CH1 contains or comprises SEQ ID NO: 9, and LC2 CL contains or comprises SEQ ID NO: 1 or 6. 75. An antibody according to any one of embodiments 26 to 74, wherein the light chain (LC) variable domain (VL) framework region (FR) and / or the heavy chain (HC) variable domain (VH) framework region (FR) includes one or more substitutions. 76. The antibody according to Embodiment 75, wherein VL FR is substituted with Q38, and / or G100, and / or G101 (EU numbering). 77. The antibody according to Embodiment 75, wherein VH FR is substituted with Q39 and / or G44 (EU numbering). 78. The antibody according to any one of embodiments 75 to 77, wherein the VH FR includes substitution with Q39 and / or G44 (EU numbering), and the VL FR includes substitution with Q38 and / or G100 and / or G101 (EU numbering). 79. The antibody according to any one of Embodiments 76 to 78, wherein the VL FR substitution in Q38 includes, or consists of, a substitution selected from the group consisting of Q38D and Q38K substitutions (EU numbering). 80. The antibody according to any one of Embodiments 76 to 79, wherein the VL FR substitution in G100 includes, or consists of, a substitution selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering). 81. The antibody according to any one of Embodiments 76 to 80, wherein the VL FR substitution in G101 includes, or consists of, a substitution selected from the group consisting of G101D and G101K substitutions (EU numbering). 82. The antibody according to any one of Embodiments 77 to 81, wherein the VH FR substitution in Q39 includes, or consists of, a substitution selected from the group consisting of Q39D and Q39K substitutions (EU numbering). 83. An antibody according to any one of Embodiments 77 to 82, wherein the VH FR substitution in G44 includes, or consists of, a substitution selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering). 84. The antibody according to any one of Embodiments 75 to 83, wherein the VH FR substitution includes or consists of a Q39K substitution, and the VL FR substitution includes or consists of a Q38D substitution. 85. The antibody according to any one of Embodiments 75 to 83, wherein the VH FR substitution includes or consists of a Q39D substitution, and the VL FR substitution includes or consists of a Q38K substitution. 86. The antibody according to any one of Embodiments 75 to 85, wherein the VH FR substitution includes or consists of a G44K substitution, and the VL FR substitution includes or consists of a G100D substitution. 87. The antibody according to any one of Embodiments 75 to 85, wherein the VH FR substitution includes or consists of a G44D substitution, and the VL FR substitution includes or consists of a G100K substitution. 88. The antibody according to any one of Embodiments 75 to 85, wherein the VH FR substitution includes or consists of a G44C substitution, and the VL FR substitution includes or consists of a G100C substitution. 89. The antibody according to any one of Embodiments 75 to 85, wherein the VH FR substitution includes or consists of a G44K substitution, and the VL FR substitution includes or consists of a G101D substitution. 90. The antibody according to any one of Embodiments 75 to 85, wherein the VH FR substitution includes or consists of a G44D substitution, and the VL FR substitution includes or consists of a G101K substitution. 91. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a Q39K substitution, the substitution of LC1 VL FR includes or consists of a Q38D substitution, the substitution of HC2 VH FR includes or consists of a Q39D substitution, and the substitution of LC2 VL FR includes or consists of a Q38K substitution. 92. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a Q39D substitution, the substitution of LC1 VL FR includes or consists of a Q38K substitution, the substitution of HC2 VH FR includes or consists of a Q39K substitution, and the substitution of LC2 VL FR includes or consists of a Q38D substitution. 93. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G100D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G100K substitution. 94. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G100K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G100D substitution. 95. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution. 96. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC2 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution. 97. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G101D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G101K substitution. 98. The antibody according to any one of Embodiments 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G101K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G101D substitution. 99. The antibody according to any one of Embodiments 75 to 98, wherein HC1 or HC2 VH contains or consists of any one of SEQ ID NOs. 53 to 64. 100. The antibody according to any one of Embodiments 75 to 99, wherein LC1 or LC2 VL contains or consists of any one of SEQ ID NOs. 65 to 80. 101. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 30, 43, 35, and 52, or Sequence IDs 35, 52, 30, and 43 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 102. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 29, 44, 36, and 51, or Sequence IDs 36, 51, 29, and 44 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 103. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 48, or Sequence IDs 33, 48, 28, and 39 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 104. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 47, or Sequence IDs 34, 47, 27, and 40 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 105. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 26, 38, 31, and 45, or Sequence IDs 31, 45, 26, and 38 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 106. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 25, 37, 32, and 46, or Sequence IDs 32, 46, 25, and 37 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 107. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 28, 41, 33, and 50, or Sequence IDs 33, 50, 28, and 41 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 108. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 27, 42, 34, and 49, or Sequence IDs 34, 49, 27, and 42 An antibody according to any one of embodiments 75 to 100, comprising or consisting of the sequence. 109. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 30, LC1 contains or comprises SEQ ID NO: 43, HC2 contains or comprises SEQ ID NO: 35, and LC2 contains or comprises SEQ ID NO: 52. 110. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 29, LC1 contains or comprises SEQ ID NO: 44, HC2 contains or comprises SEQ ID NO: 36, and LC2 contains or comprises SEQ ID NO: 51. 111. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 28, LC1 contains or comprises SEQ ID NO: 39, HC2 contains or comprises SEQ ID NO: 33, and LC2 contains or comprises SEQ ID NO: 48. 112. The antibody according to any one of embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 27, LC1 contains or comprises SEQ ID NO: 40, HC2 contains or comprises SEQ ID NO: 34, and LC2 contains or comprises SEQ ID NO: 47. 113. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 26, LC1 contains or comprises SEQ ID NO: 38, HC2 contains or comprises SEQ ID NO: 31, and LC2 contains or comprises SEQ ID NO: 45. 114. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 25, LC1 contains or comprises SEQ ID NO: 37, HC2 contains or comprises SEQ ID NO: 32, and LC2 contains or comprises SEQ ID NO: 46. 115. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 28, LC1 contains or comprises SEQ ID NO: 41, HC2 contains or comprises SEQ ID NO: 33, and LC2 contains or comprises SEQ ID NO: 50. 116. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 27, LC1 contains or comprises SEQ ID NO: 42, HC2 contains or comprises SEQ ID NO: 34, and LC2 contains or comprises SEQ ID NO: 49. 117. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 35, LC1 contains or comprises SEQ ID NO: 52, HC2 contains or comprises SEQ ID NO: 30, and LC2 contains or comprises SEQ ID NO: 43. 118. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 36, LC1 contains or comprises SEQ ID NO: 51, HC2 contains or comprises SEQ ID NO: 29, and LC2 contains or comprises SEQ ID NO: 44. 119. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 33, LC1 contains or comprises SEQ ID NO: 48, HC2 contains or comprises SEQ ID NO: 28, and LC2 contains or comprises SEQ ID NO: 39. 120. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 34, LC1 contains or comprises SEQ ID NO: 47, HC2 contains or comprises SEQ ID NO: 27, and LC2 contains or comprises SEQ ID NO: 40. 121. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 31, LC1 contains or comprises SEQ ID NO: 45, HC2 contains or comprises SEQ ID NO: 26, and LC2 contains or comprises SEQ ID NO: 38. 122. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 32, LC1 contains or comprises SEQ ID NO: 46, HC2 contains or comprises SEQ ID NO: 25, and LC2 contains or comprises SEQ ID NO: 37. 123. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 33, LC1 contains or comprises SEQ ID NO: 50, HC2 contains or comprises SEQ ID NO: 28, and LC2 contains or comprises SEQ ID NO: 41. 124. The antibody according to any one of Embodiments 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 34, LC1 contains or comprises SEQ ID NO: 49, HC2 contains or comprises SEQ ID NO: 27, and LC2 contains or comprises SEQ ID NO: 42. 125. A composition comprising one or more polynucleotides encoding the Fab and / or antibody described in any one of Embodiments 1 to 124. 126. The composition according to Embodiment 125, further comprising one or more polynucleotides encoding an IgG Fab or antibody. 127. An expression vector or construct for the expression of one or more polynucleotides as described in any one of Embodiments 125 to 126. 128. An expression vector or construct according to Embodiment 127, wherein the expression vector is a plasmid or a viral vector. 129. A cell comprising one or more polynucleotides, expression vectors, or constructs described in any one of Embodiments 125 to 128. 130. A cell expressing one or more polynucleotides encoding the Fab and / or antibody or a fragment thereof as described in any one of Embodiments 1 to 124. 131. Cells are mammalian cells or A bacterial cell, as described in any one of embodiments 129 to 130. 132. The cell according to any one of Embodiments 129 to 131, wherein the cell is HEK293 cell, Chinese hamster ovary (CHO) cell, or NS0 cell. 133. A pharmaceutical composition comprising the Fab and / or antibody or fragment thereof described in any one of Embodiments 1 to 124. 134. A composition comprising cells according to any one of embodiments 129 to 132, wherein the composition is optionally a cell culture. 135. A composition comprising the cell supernatant and / or cell lysate of the cells described in any one of Embodiments 129 to 132. 136. A composition comprising an antibody comprising two heavy / light chain pairs selected from the HC1 / LC1, HC2 / LC2, HC1 / LC2 and HC2 / LC1 pairs described in any one of Embodiments 1 to 124, wherein at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the antibody comprises an HC1 / LC1 pair and an HC2 / LC2 pair. 137. The composition according to Embodiment 136, wherein the composition does not contain an antibody comprising either HC1 / LC2 or HC2 / LC1, or contains 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less of such antibody. 138. The composition according to any one of Embodiments 133 to 137, wherein the composition is a cell culture supernatant and / or cell lysate, and the composition contains at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of antibodies containing HC1 / LC1 pairs and HC2 / LC2 pairs before any concentration of the composition for antibodies containing HC1 / LC1 pairs and HC2 / LC2 pairs. 139. The composition according to Embodiment 138, wherein the composition does not contain an antibody or fragment thereof containing either an HC1 / LC2 pair or an HC2 / LC1 pair, or contains 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less, prior to any concentration of the composition for an antibody containing an HC1 / LC1 pair and an HC2 / LC2 pair. 140. A method for producing an antibody or a fragment thereof, the method being: The present invention provides a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL). HC1 CH1 includes replacement with F170 and S183, and replacement with V185 (EU numbering), LC1 CL includes replacement with L135 (EU numbering), HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). LC1 CL is a method that does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 141. The method according to Embodiment 140, wherein HC1 CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and LC1 CL consists of substitutions in L135 (EU numbering). 142. A method for producing an antibody or fragment thereof according to Embodiment 141, further comprising substitution of HC1 CH1 with V185. 143. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 142, wherein the HC1 CH1 substitution in F170 (EU numbering) is an F170I or F170V substitution. 144. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 143, wherein the HC1 CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. 145. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 144, wherein the HC1 CH1 substitution in V185 (EU numbering) is a V185L substitution. 146. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 145, wherein the LC1 CL1 substitution in L135 (EU numbering) is an L135F substitution. 147. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 146, wherein HC1 CH1 consists of F170I and S183V (EU numbering) substitutions, and LC1 CL consists of L135F (EU numbering) substitution. 148. A method for producing an antibody or fragment thereof according to any one of Embodiments 140 to 147, wherein HC1 CH1 consists of F170I, S183L, and V185L (EU numbering) substitutions, and LC1 CL consists of L135F (EU numbering) substitution. 149. A method for producing an antibody or a fragment thereof, wherein the method is: The invention includes providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, Each HC1 and HC2 contains the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1). Each LC1 and LC2 contains the variable light chain domain (VL) and constant light chain domain (CL) of human IgG. HC1 CH1 includes or consists of substitutions for F126, F170, S183, V185, and C220 (EU numbering). LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and optionally LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) (EU numbering), or optionally LC1 CL includes or consists of substitutions for Q124, L135, and C214 (kappa) (EU numbering), HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). LC1 CL is a method that does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 150. The method according to Embodiment 149, wherein the substitution of HC1 CH1 in F126 (EU numbering) is the substitution of F126C. 151. The method according to any one of Embodiments 149 to 150, wherein the HC1 CH1 substitution in F170 (EU numbering) is F170V or F170I substitution. 152. The method according to any one of embodiments 149 to 151, wherein the HC1 CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. 153. The method according to any one of Embodiments 149 to 152, wherein the substitution of HC1 CH1 in V185 (EU numbering) is the substitution of V185L. 154. The method according to any one of embodiments 149 to 153, wherein the substitution of HC1 CH1 in C220 (EU numbering) is C220S substitution. 155. The method according to any one of embodiments 149 to 154, wherein the LC1 CL substitution at position 124 (EU numbering) is E124C for lambda or Q124C for kappa LC, and optionally, the LC1 CL substitution at position 124 (EU numbering) is E124C for lambda LC, or optionally, the LC1 CL substitution at position 124 (EU numbering) is Q124C for kappa LC. 156. The method according to Embodiments 149 to 155, wherein the LC1 CL substitution in L135 (EU numbering) is the L135F substitution. 157. The method according to embodiments 149 to 156, wherein the LC1 CL substitution in C214 (EU numbering) is C214S substitution. 158. The method according to Embodiment 149, wherein HC1 CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC1 CL includes or consists of substitutions for E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally including or consisting of substitutions for E124C, L135F, and C214S (lambda) (EU numbering), or optionally including or consisting of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). 159. The method according to Embodiment 149, wherein HC1 CH1 includes or consists of the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC1 CL includes or consists of the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally including or consisting of the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally including or consisting of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering). 160. A method for producing an antibody or a fragment thereof, wherein the method is: The present invention provides a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL). HC1 CH1 includes or consists of substitutions to F126 and C220 (EU numbering), LC1 CL includes or consists of substitutions to E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering) (optionally to E124 and C214 (lambda), or optionally to Q124 and C214 (kappa)), HC2 CH1 includes or consists of substitutions to F170, S183, and V185 (EU numbering), LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 do not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), LC1 CL and / or LC2 CL are methods that do not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). 161. The method according to Embodiment 160, wherein the HC1 CH1 substitution in F126 (EU numbering) is F126C substitution, the LC1 CL substitution in position 124 (EU numbering) is E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC1 CL substitution in position 124 (EU numbering) is E124C (lambda) substitution, or optionally, the LC1 CL substitution in position 124 (EU numbering) is Q124C (kappa) substitution. 162. The method according to any one of embodiments 160 to 161, wherein the substitution of HC1 CH1 in C220 (EU numbering) is C220S substitution. 163. The antibody according to any one of embodiments 160 to 162, wherein the LC1 CL substitution in C214 (EU numbering) is a C214S substitution. 164. The method according to any one of embodiments 160 to 163, wherein the substitution of HC2 CH1 in F170 (EU numbering) is F170V or F170I substitution. 165. The method according to any one of embodiments 160 to 164, wherein the HC2 CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. 166. The method according to any one of embodiments 160 to 165, wherein the substitution of HC2 CH1 in V185 (EU numbering) is V185L substitution. 167. The method according to any one of embodiments 160 to 166, wherein the LC2 CL substitution in L135 (EU numbering) is an L135F substitution. 168. The method according to Embodiment 160, wherein HC1 CH1 includes or consists of the substitutions F126C and C220S (EU numbering), LC1 CL includes or consists of the substitutions E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) (optionally, LC1 CL includes or consists of the substitutions E124C and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitutions Q124C and C214S (kappa) (EU numbering)), HC2 CH1 includes or consists of F170I, S183L, V185L (EU numbering), and LC2 CL includes or consists of the substitution L135F (EU numbering). 169. The method according to Embodiment 160, wherein HC1 CH1 includes or consists of the substitution of F126C and C220S (EU numbering), LC1 CL includes or consists of the substitution of E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) (optionally, LC1 CL includes or consists of the substitution of E124C and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitution of Q124C and C214S (kappa) (EU numbering)), HC2 CH1 includes or consists of F170V, S183I, V185L (EU numbering), and LC2 CL includes or consists of the substitution of L135F (EU numbering). 170. The method according to any one of Embodiments 140 to 169, wherein an antibody comprising two heavy / light chain pairs is produced, the first pair being HC1 / LC1 and the second pair being HC2 / LC2. 171. The method according to any one of Embodiments 140 to 170, wherein the antibody further comprises an HC1 / HC2 pair. 172. The method according to embodiment 171, wherein HC1 and HC2 are paired via charge-charge interaction. 173. The method according to any one of embodiments 140 to 172, wherein each HC1 and HC2 further comprises a heavy chain constant domain (CH3), and the HC1 and HC2 CH3 together constitute a knob-into-hole structure. 174. The method according to embodiment 173, wherein HC1 CH3 and / or HC2 CH3 constitute a knob and HC2 CH3 constitutes a hole. 175. The method according to embodiment 173, wherein HC1 CH3 constitutes a hole and HC2 CH3 constitutes a knob. 176. The method according to any one of embodiments 171 to 175, wherein HC1 CH3 and / or HC2 CH3 include, or consist of, substitutions of T366, L368, and Y407 (EU numbering). 177. The method according to any one of embodiments 171 to 176, wherein the substitution of HC1 CH3 and / or HC2 CH3 in T366 (EU numbering) is a substitution of T366W or T366S. 178. The method according to any one of embodiments 171 to 177, wherein the substitution of HC1 CH3 and / or HC2 CH3 in L368 (EU numbering) is the substitution of L368A. 179. The method according to any one of embodiments 171 to 178, wherein the substitution of HC1 CH3 and / or HC2 CH3 in Y407 (EU numbering) is Y407V substitution. 180. The method according to any one of embodiments 171 to 179, wherein HC1 CH3 includes or consists of the T366W (EU numbering) substitution, and HC2 CH3 includes or consists of the T366S, L368A, and Y407V (EU numbering) substitutions. 181. The method according to any one of embodiments 171 to 180, wherein HC1 CH3 includes or consists of T366S, L368A, and Y407V (EU numbering) substitutions, and HC2 CH3 includes or consists of T366W (EU numbering) substitution. 182. The method according to any one of embodiments 140 to 181, wherein LC1 and / or LC2 are kappa light chains. 183. The method according to any one of embodiments 140 to 181, wherein LC1 and / or LC2 are lambda light chains. 184. The method according to any one of embodiments 140 to 181, wherein LC1 is a lambda light chain and LC2 is a kappa light chain. 185. The method according to any one of embodiments 140 to 181, wherein LC1 is a kappa light chain and LC2 is a lambda light chain. 186. The method according to any one of Embodiments 140 to 185, wherein VL FR is replaced with Q38, and / or G100, and / or G101 (EU numbering). 187. The method according to any one of Embodiments 140 to 186, wherein VH FR is replaced with Q39 and / or G44 (EU numbering). 188. The method according to any one of Embodiments 140 to 187, wherein VH FR includes substitution with Q39 and / or G44 (EU numbering), and VL FR includes substitution with Q38 and / or G100 and / or G101 (EU numbering). 189. The method according to any one of Embodiments 140 to 188, wherein the VL FR substitution in Q38 includes, or consists of, substitutions selected from the group consisting of Q38D and Q38K substitutions (EU numbering). 190. The method according to any one of Embodiments 140 to 188, wherein the VL FR substitution in G100 includes, or consists of, substitutions selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering). 191. The method according to any one of Embodiments 140 to 188, wherein the VL FR substitution in G101 includes, or consists of, a substitution selected from the group consisting of G101D and G101K substitutions (EU numbering). 192. The method according to any one of Embodiments 140 to 188, wherein the VH FR substitution in Q39 includes, or consists of, substitutions selected from the group consisting of Q39D and Q39K substitutions (EU numbering). 193. The method according to any one of Embodiments 140 to 188, wherein the VH FR substitution in G44 includes, or consists of, substitutions selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering). 194. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a Q39K substitution, and the substitution of VL FR includes or consists of a Q38D substitution. 195. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a Q39D substitution, and the substitution of VL FR includes or consists of a Q38K substitution. 196. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G100D substitution. 197. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G100K substitution. 198. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a G44C substitution, and the substitution of VL FR includes or consists of a G100C substitution. 199. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G101D substitution. 200. The method according to any one of Embodiments 140 to 193, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G101K substitution. 201. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a Q39K substitution, the substitution of LC1 VL FR includes or consists of a Q38D substitution, the substitution of HC2 VH FR includes or consists of a Q39D substitution, and the substitution of LC2 VL FR includes or consists of a Q38K substitution. 202. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a Q39D substitution, the substitution of LC1 VL FR includes or consists of a Q38K substitution, the substitution of HC2 VH FR includes or consists of a Q39K substitution, and the substitution of LC2 VL FR includes or consists of a Q38D substitution. 203. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G100D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G100K substitution. 204. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G100K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G100D substitution. 205. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution. 206. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC2 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution. 207. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G101D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G101K substitution. 208. The method according to any one of Embodiments 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G101K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G101D substitution. 209. The method according to any one of Embodiments 140 to 208, wherein HC1 or HC2 VH includes or consists of any one of Sequence IDs 53 to 64. 210. The method according to any one of Embodiments 140 to 209, wherein LC1 or LC2 VL includes or consists of any one of Sequence IDs 65 to 80. 211. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 30, 43, 35, and 52, or Sequence IDs 35, 52, 30, and 43 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 212. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 29, 44, 36, and 51, or Sequence IDs 36, 51, 29, and 44 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 213. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 48, or Sequence IDs 33, 48, 28, and 39 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 214. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 47, or Sequence IDs 34, 47, 27, and 40 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 215. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 26, 38, 31, and 45, or Sequence IDs 31, 45, 26, and 38 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 216. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 25, 37, 32, and 46, or Sequence IDs 32, 46, 25, and 37 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 217. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 50, or Sequence IDs 33, 50, 28, and 41 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 218. Each of the heavy and light chains of the HC1 / LC1 pair and the heavy and light chains of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 49, or Sequence IDs 34, 49, 27, and 42 The method according to any one of embodiments 140 to 210, comprising or consisting of an array of . 219. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 30, LC1 includes or consists of sequence number 43, HC2 includes or consists of sequence number 35, and LC2 includes or consists of sequence number 52. 220. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 29, LC1 includes or consists of sequence number 44, HC2 includes or consists of sequence number 36, and LC2 includes or consists of sequence number 51. 221. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 48. 222. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 47. 223. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 26, LC1 includes or consists of sequence number 38, HC2 includes or consists of sequence number 31, and LC2 includes or consists of sequence number 45. 224. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 25, LC1 includes or consists of sequence number 37, HC2 includes or consists of sequence number 32, and LC2 includes or consists of sequence number 46. 225. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 41, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 50. 226. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 42, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 49. 227. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 35, LC1 includes or consists of sequence number 52, HC2 includes or consists of sequence number 30, and LC2 includes or consists of sequence number 43. 228. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 36, LC1 includes or consists of sequence number 51, HC2 includes or consists of sequence number 29, and LC2 includes or consists of sequence number 44. 229. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 48, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 39. 230. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 47, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 40. 231. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 31, LC1 includes or consists of sequence number 45, HC2 includes or consists of sequence number 26, and LC2 includes or consists of sequence number 38. 232. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 32, LC1 includes or consists of sequence number 46, HC2 includes or consists of sequence number 25, and LC2 includes or consists of sequence number 37. 233. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 50, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 41. 234. The method according to any one of embodiments 201 to 218, wherein HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 49, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 42. 235. The method according to any one of Embodiments 140 to 234, wherein multiple antibodies containing two heavy / light chain pairs are produced, and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the produced antibodies containing two heavy / light chain pairs contain HC1 / LC1 and HC2 / LC2. 236. The method according to any one of Embodiments 140 to 235, wherein 0% or less of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the antibody containing two heavy / light chain pairs produced contains either HC1 / LC2 or HC2 / LC1. 237. The method according to Embodiment 236, wherein the percentage (%) of the antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs refers to the percentage of HC1 / LC1 pairs and HC2 / LC2 pairs of the antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs before any enrichment. 238. The method according to Embodiment 236, wherein the percentage (%) of the antibody containing HC1 / LC2 pairs and HC2 / LC1 pairs refers to the percentage of HC1 / LC2 pairs and HC2 / LC1 pairs of the antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs before any enrichment. 239. The method according to any one of Embodiments 140 to 238, comprising providing one or more polynucleotides encoding HC1, HC2, LC1, and LC2. 240. The method according to Embodiment 239, wherein one or more polynucleotides encode LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or in an approximately ratio, optionally 1:5 or approximately 1:5. 241. The method according to Embodiment 240, wherein one or more polynucleotides encode HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately in ratio. 242. The method according to Embodiment 241, wherein one or more polynucleotides encode HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately in ratio. 243. The method according to Embodiment 239, wherein one or more polynucleotides encode HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately in ratio. 244. The method according to any one of embodiments 239 to 243, wherein one or more polynucleotides encode an IgG Fab or an antibody. 245. The method according to any one of Embodiments 141 to 239, comprising providing one or more expression vectors or constructs encoding HC1, HC2, LC1, and LC2. 246. The method according to Embodiment 245, wherein one or more expression vectors or constructs encode LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately, and optionally the ratio is 1:5 or approximately 1:5. 247. The method according to Embodiment 246, wherein one or more expression vectors or constructs encode HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately in a ratio. 248. The method according to Embodiment 247, wherein one or more expression vectors or constructs encode HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately in ratio. 249. The method according to Embodiment 245, wherein one or more expression vectors or constructs encode HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately in ratio. 250. The method according to any one of embodiments 245 to 249, wherein one or more expression vectors are plasmids or viral vectors. 251. The method according to any one of Embodiments 245 to 250, wherein one or more expression vectors or constructs encode an IgG Fab or an antibody. 252. The method according to any one of Embodiments 140 to 251, comprising providing cells expressing HC1, HC2, LC1 and LC2. 253. The method according to Embodiment 252, wherein the cells express LC1 and LC2 in a ratio of 8:1 to 1:8, optionally in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately in a ratio, optionally in a ratio of 1:5 or approximately 1:5. 254. The method according to Embodiment 253, wherein the cells express HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately in that ratio. 255. The method according to Embodiment 254, wherein the cells express HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately in that ratio. 256. The method according to Embodiment 252, wherein the cells express HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately in that ratio. 257. The method according to any one of embodiments 252 to 256, wherein cells express IgG Fab or an antibody. 258. The method according to any one of embodiments 252 to 257, wherein the cells are mammalian cells or bacterial cells. 259. The method according to any one of Embodiments 252 to 257, wherein the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells. 260. The method according to any one of Embodiments 140 to 259, further comprising extraction and / or purification of an antibody or a fragment thereof. 261. The method according to Embodiment 260, wherein an antibody or a fragment thereof is extracted and / or purified from cell supernatant or cell lysate. 262. The Fab according to any one of Embodiments 1 to 30, wherein the Fab is a chimeric Fab, a humanized Fab, or a human Fab. 263. An antibody, composition, cell, expression vector or construct, polynucleotide, or method according to any one of Embodiments 31 to 261, wherein the antibody comprises or consists of an IgG antibody. 264. The antibody, composition, cell, expression vector or construct, polynucleotide, or method according to Embodiment 263, wherein the IgG antibody is IgG1, IgG2, IgG3, or IgG4. 265. An antibody, composition, cell, expression vector or construct, polynucleotide, or method according to any one of embodiments 263 to 264, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody. 266. Any one of embodiments 1 to 265, wherein the fragment comprises an antigen-binding fragment. [Brief explanation of the drawing]

[0006] [Figure 1] This is a diagram of an embodiment of an antigen-binding fragment (Fab). [Figure 2] This is a diagram illustrating an embodiment of the antibody. [Figure 3] This is a flowchart of an embodiment of a method for screening for compensatory mutations in the CL and CH1 domains that transmit LC and HC specific pairing for an antibody or its fragment. [Figure 4] This is an exemplary representation of an embodiment in which a light chain having L135F is used to interfere with the correct pairing of the WT heavy chain to the WT light chain. [Figure 5] This is an exemplary representation of an embodiment in which a wt light chain is used to interfere with the correct pairing of a mutant heavy chain to a mutant light chain. [Figure 6A] This table shows the identity of the sample embodiments in each lane of the SDS-page gel in Figure 6B, using the screening strategies shown in Figures 4 and 5. [Figure 6B]These are SDS-PAGE gels representing different embodiments of HC / LC pairing under non-reducing conditions. Mutation sets that enhance LC / HC pairing specificity enhance the percentage of correctly assembled intact antibodies (upper bands of two LCs and two HCs) in the presence of similar amounts of competing, unchanged wt LC-JT7 or 11 / LC-F pairing, and achieve equivalent correct assembly of wt IgG in the presence of competing wt LCs. [Figure 7A] This table shows the identity of the sample embodiments in each lane of the SDS-page gel in Figure 7B, using the screening strategies shown in Figures 4 and 5. [Figure 7B] These are SDS-page gels representing different embodiments of HC / LC pairing under reducing conditions. [Figure 8] These are a series of chromatograms illustrating embodiments of RP-HPLC quantification of correct LC-HC pairings in positive controls and selected mutant sets. [Figure 9A] This is an illustrative representation of the correct pairing of the light chain with the L135F mutation with the dimerizing heavy chains of JT7 and JT11 in an example of a bispecific antibody (bsMab). [Figure 9B] This is a chromatogram of an embodiment of purification and correct assembly analysis by RP-HPLC for purified bispecific antibodies using an LC / HC pairing strategy for selective disulfide (R2), JT7 / F, and JT11 / F dimerized pairs. [Figure 9C] This is an SDS-page gel of an embodiment of a bispecific antibody (LC-HC dimer) correctly assembled under reducing and non-reducing conditions. [Figure 10A] This graph shows an embodiment of the neutralizing activity of the bispecific antibody (bsMab) in Fab arm 1 of a cytokine A blockade assay using a bispecific antibody in HEK-293 reporter cells. [Figure 10B] This graph shows an embodiment of the neutralizing activity of the bispecific antibody (bsMab) in Fab arm 2 of a cytokine B blockade assay using a bispecific antibody in HEK-293 reporter cells. [Figure 11A]This is an SDS-page gel of an embodiment of bsMab expressing R2, R2+JT7 / F, and R2+JT11 / F in 293 cells under reducing and non-reducing conditions. [Figure 11B] Figure 11C shows a table quantifying the yield of expression by ProA capture, and a table showing the percentage of correct assembly in the ProA pool analyzed by RP-HPLC. [Figure 11C] This is a set of chromatograms representing embodiments of RP-HPLC quantification of correct LC-HC pairing of bsMab expressed in R2, R2+JT7 / F, and R2+JT11 / F cells in the protein A pool. [Figure 12] This is a table showing embodiments of the CL sequence in the wild-type and mutant CL domains of lambda and kappa LC. [Figure 13-1] This is a table showing embodiments of the CH1 sequence in the wild-type and mutant CH1 domains of IGHG1. [Figure 13-2] This is a table showing embodiments of the CH1 sequence in the wild-type and mutant CH1 domains of IGHG1. [Figure 14-1] This table shows several embodiments of sequence alignments for the wild-type and mutant CH1 domains of IGGH1. Blank residues indicate consensus for IGGH1 CH1. Substituted residues are indicated at each position as shown in the figure. [Figure 14-2] This table shows several embodiments of sequence alignments for the wild-type and mutant CH1 domains of IGGH1. Blank residues indicate consensus for IGGH1 CH1. Substituted residues are indicated at each position as shown in the figure. [Figure 14-3] This table shows several embodiments of sequence alignments for the wild-type and mutant CH1 domains of IGGH1. Blank residues indicate consensus for IGGH1 CH1. Substituted residues are indicated at each position as shown in the figure. [Figure 15-1]This table shows several embodiments of sequence alignments for the lambda and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 15-2] This table shows several embodiments of sequence alignments for the lambda and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 15-3] This table shows several embodiments of sequence alignments for the lambda and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 16-1] This table shows several embodiments of sequence alignments for the kappa and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 16-2] This table shows several embodiments of sequence alignments for the kappa and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 16-3] This table shows several embodiments of sequence alignments for the kappa and mutant CL domains. Blank residues indicate consensus for lambda CL. Substituted residues are indicated at each position as shown in the figure. [Figure 17A] This bar graph illustrates a method for quantifying bispecific antibody expression in CHO cells. [Figure 17B] This bar graph shows an embodiment of the quantification of the ProA pool analyzed by capillary electrophoresis sodium dodecyl sulfate (CE-SDS). [Figure 17C] This bar graph illustrates an example of quantifying mismatch species using mass spectrometry. [Figure 18A]This is a chromatogram illustrating an embodiment of quantification of Fab fragment pairings analyzed by LC-MS. [Figure 18B] This chromatogram shows an embodiment of the quantification of purified Fab fragment pairings, analyzed by LC-MS. Detailed description of the invention

[0007] The following detailed description refers to the accompanying drawings which form part of this specification. In the drawings, similar symbols usually indicate similar components unless the context specifically indicates otherwise. The exemplary embodiments described in the detailed description, drawings and claims are not intended to be limiting. Other embodiments may be utilized and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that the aspects of this disclosure described herein and shown in the drawings can be arranged, substituted, combined, separated and designed in a wide variety of different configurations, all of which are expressly intended herein.

[0008] Unless otherwise specified, all technical and scientific terms used herein, when read in light of this specification, have the same meaning as those generally understood by those skilled in the art to which the claimed subject matter belongs. The above general description and the following detailed descriptions are illustrative and explanatory only and should not be considered limiting to any claimed subject matter.

[0009] The chapter headings used herein are for organizational purposes only and should not be interpreted as limiting the subjects covered.

[0010] For example, several approaches have been established for producing antibodies having two different antigen-binding fragment (Fab) arms, such as in International Publication No. 2021155461. However, the indiscriminate pairing of antibody heavy chains (HC) and light chains (LC) remains a challenge for their efficient production. Therefore, some embodiments herein focus on Fabs, antibodies, and their fragments with improved heavy chain HC-HC and HC-LC pairing efficiency. Methods for producing Fabs, antibodies, and their fragments with improved heavy chain HC-HC and HC-LC pairing efficiency are also disclosed herein.

[0011] For example, some embodiments herein relate to antigen-binding fragments (Fab) comprising a heavy chain variable domain (VH), a heavy chain constant domain (CH1), a light chain variable domain (VL), and a light chain constant domain (CL), wherein HC CH1 consists of substitutions at F170, S183, and V185, and LC1 CL consists of substitutions at L135 (EU numbering).

[0012] Some embodiments herein relate to antibodies or fragments thereof comprising a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, where each HC1 and HC2 comprises a heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 consists of substitutions at F170, S183, and V185, and LC1 CL consists of a substitution at L135 (EU numbering). In some embodiments, the fragment comprises an antigen-binding fragment.

[0013] Some embodiments herein relate to methods for producing antibodies or fragments thereof, the methods comprising providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprising a light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 consists of substitutions at F170, S183, and V185, and LC1 CL consists of a substitution at L135 (EU numbering). In some embodiments, the fragment comprises an antigen-binding fragment.

[0014] term The articles "a" and "an," when read in accordance with this specification, have their plain, ordinary meanings and refer to one or more (e.g., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0015] Throughout this specification, unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising,” when read in light of this specification, will be understood to have their plain, ordinary meanings, meaning to include the described process or element, or group of processes or elements, but not to exclude any other process or element, or group of processes or elements. “Consists of” means to include and be limited to everything that follows the phrase “consists of.” Thus, the phrase “consists of” indicates that the enumerated elements are necessary or essential, and other elements may not be present. “Essentially consists of” means to include any elements enumerated after the phrase, and be limited to other elements that do not interfere with or contribute to the activity or action of the enumerated elements as specified in this disclosure. Thus, the phrase “essentially consists of” indicates that the enumerated elements are necessary or essential, but other elements are optional and may or may not be present, depending on whether they substantially affect the activity or action of the enumerated elements.

[0016] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and have their plain, ordinary meanings when read in light of this specification, including references to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, and may contain modified amino acids or be interrupted by non-amino acids. The term also encompasses amino acid polymers modified through any other operation, such as sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodization, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, arginylation, transfer RNA-mediated addition of amino acids to proteins, ubiquitination, or conjugation with labeling components.

[0017] As used herein, the term “amino acid” has its plain, ordinary meaning when read in light of this specification and refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimetic substances.

[0018] A polypeptide or amino acid sequence "derived from" a specified protein has its plain, ordinary meaning when read in light of this specification and refers to the origin of the polypeptide. Preferably, the polypeptide has an amino acid sequence, or a portion thereof, that is essentially identical to the amino acid sequence of the polypeptide encoded in the sequence, the portion consisting of at least 10-20 amino acids, or at least 20-30 amino acids, or at least 30-50 amino acids, or immunologically identifiable with the polypeptide encoded in the sequence. This term also includes polypeptides expressed from a specified nucleic acid sequence. A peptide sequence is assumed to have at least 80%, 85%, 90%, 95%, 99%, or 100% homology with any one of the peptide sequences disclosed herein and to have the same or similar functional properties. The homology percentage may be determined according to amino acid substitutions, deletions, or additions between the two peptide sequences. Peptide sequences having some percentage homology with any one of the peptide sequences disclosed herein can be produced and tested by those skilled in the art by conventional methods. The % homology or % identity of two sequences is well understood in the art and can be calculated by the number of conserved amino acids or nucleotides relative to the length of the sequence.

[0019] As used herein, the terms “antibody” or “immunoglobulin” have their plain, ordinary meanings when read in light of this specification, and represent the meanings attributed thereto to those skilled in the art. Furthermore, they are intended to include any polypeptide chain-containing molecular structure having a specific shape that fits and recognizes an epitope, and one or more non-covalent interactions that stabilize the complex between the molecular structure and the epitope. The antibodies used in the disclosed embodiments may be polyclonal antibodies, but monoclonal antibodies are preferred because monoclonal antibodies can be regenerated by cell culture or recombination and can be modified to reduce their antigenicity.

[0020] Spontaneous immunoglobulins are tetrameric glycoproteins, each tetramer containing two identical polypeptide chain pairs, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region consisting of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminus of each chain defines a constant region, primarily responsible for effector function. The amino-terminus of the polypeptide chain exhibits considerable variation in amino acid composition and is called the variable heavy chain (VH) and variable light chain (VL) regions, distinguishing them from the relatively constant heavy chain (CH) and constant light chain (CL) regions. Each LC consists of one VL and one CL. HC consists of the VH and three constant heavy chain domains CH1, CH2, and CH3. Antibodies can be functionally separated into VL and VH domains that bind to the antigen, and CL and CH domains that identify effector functions such as complement activation or binding to the Fc receptor. Each variable domain can be divided into three variable sequence regions called complementarity-determining regions or CDRs, and four relatively constant sequence regions called framework regions or FRs. The three CDRs of HC pair with the three CDRs of LC, as classically defined, to form the antigen-binding site. There are five major classes of HC and CH domains. Each class defines the isotypes IgM, IgG, IgA, IgD, and IgE. IgG can be subdivided into four subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biological properties, and IgA can similarly be subdivided into IgA1 and IgA2. The constant domains of HC can be switched to allow modification of effector function while maintaining antigen specificity.

[0021] As used herein, the term “chimeric antibody” has its plain, ordinary meaning when read in light of this specification and refers to an antibody or fragment thereof containing a variable domain of an antibody from one species and a constant domain of an antibody from a different species. “Humanized antibody” has its plain, ordinary meaning when read in light of this specification and refers to an antibody or fragment thereof containing a variable or constant domain of a human antibody and a variable or constant domain of an antibody from a different species. In some embodiments, the fragment includes an antigen-binding fragment.

[0022] In addition to whole immunoglobulins (or their recombinant counterparts), immunoglobulin fragments or "binding fragments" containing epitope-binding sites (e.g., Fab', F(ab')2, single-chain variable fragments (scFv), diabodies, minibodies, nanobodies, single-domain antibodies (sdAb), or other fragments) are useful as antibody portions in the disclosed embodiments. Such antibody fragments may be generated from whole immunoglobulins by cleavage with lysine, pepsin, papain, or other proteases. Miniature immunoglobulins may be designed using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the disclosed embodiments may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., polyglycine, or another sequence that does not form an α-helix or β-sheet motif). Nanobodies or single-domain antibodies may also be derived from selective organisms such as dromedary camels, camels, llamas, alpacas, or sharks. In some embodiments, the antibody may be a conjugate, such as a pegylated antibody, drug, radioisotope, or toxin conjugate. Monoclonal antibodies directed against a specific epitope or combination of epitopes allow for the targeting and / or depletion of cell populations expressing the marker. A variety of techniques can be utilized to screen cell populations expressing the marker using monoclonal antibodies, including magnetic separation using antibody-coated magnetic beads, "panning" using antibodies attached to a solid matrix (i.e., a plate), and flow cytometry (e.g., U.S. Patent No. 5,985,660, which is expressly incorporated herein by reference in its entirety).

[0023] As used herein, the term “Fc region” has its plain, ordinary meaning when read in light of this specification and refers to the C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a mutant Fc region. While the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus. The numbering of residues in the Fc region follows the EU index, as in Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. As is known in the art, the Fc region may exist in dimeric or monomeric form.

[0024] In some embodiments, the definitive demarcation of the CDR and the identification of residues containing antibody binding sites are achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, the CDR region can be identified or approximated using various analytical methods. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the IMGT approach (Lefranc et al., 2003, Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), the AbM definition, and the conformational definition.

[0025] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDRs and other antibody regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but takes into account the location of specific structural loop regions. See, for example, Chothia et al., 1986, J.Mol.Biol., 196:901-17 and Chothia et al., 1989, Nature, 342:877-83. The AbM definition uses an integrated suite of computer programs developed by the Oxford Molecular Group to model antibody structures. For example, see Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272, "AbM.TM., A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. The AbM definition involves modeling the tertiary structure of an antibody from its primary sequence using a combination of knowledge databases and the ab initio method, such as those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The definition of contact is based on the analysis of the crystal structure of available complexes. For example, see MacCallum et al., 1996, J.Mol.Biol., 5:73245.As used herein, CDR, Fab fragment, CL, CH1, CH2, CH3, VL, and / or VH have their plain, ordinary meanings when read in light of this specification and may refer to CDR, Fab fragment, CL, CH1, CH2, CH3, VL, and / or VH as defined by any approach known in the art, including combinations of approaches. Methods used herein may utilize CDR, CL, CH1, CH2, CH3, VL, and / or VH as defined according to any of these approaches. In any given embodiment including two or more CDR, Fab, CL, CH1, CH2, CH3, VL, and / or VH, CDR, Fab, CL, CH1, CH2, CH3, VL, and / or VH may be defined according to any of the definitions of Kabat, Chothia, extension, IMGT, Paratome, AbM, and / or conformation, or any combination thereof.

[0026] As used herein, the term “constant region” with respect to an antibody has its plain, ordinary meaning when read in light of this specification, and refers to either the constant region of an antibody light chain or the constant region of an antibody heavy chain, either alone or in combination.

[0027] The “variable region” of an antibody, as read herein, has its plain, ordinary meaning and refers to either the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, and contribute to the formation of the antibody's antigen-binding site. When a variant of the target variable region, particularly one having substitutions in amino acid residues outside the CDR region (i.e., the framework region), is desired, appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the target variable region with the variable region of other antibodies containing CDR1 and CDR2 sequences in the same canonical class as the target variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0028] As used herein, the term “antigen-binding molecule” has its plain, ordinary meaning when read in light of this specification and refers to a molecule comprising an antigen-binding moiety that binds to an antigen, and optionally a scaffold or framework moiety that facilitates the binding of the antigen-binding moiety or allows the antigen-binding moiety to incorporate conformations that provide certain additional properties to the antigen-binding molecule. In some embodiments, the antigen-binding moiety comprises at least one CDR derived from an antibody that binds to the antigen. In some embodiments, the antigen-binding moiety comprises all three CDRs derived from the heavy chain or light chain of the antibody that binds to the antigen. In some embodiments, the antigen-binding moiety comprises all six CDRs derived from the antibody that binds to the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding moiety is an antibody fragment.

[0029] Non-exclusive examples of antigen-binding molecules include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody analogs. Further specific examples include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., VH domains and VHH fragments of camelid heavy-chain antibodies; see Cortez-Retamozo et al., Cancer Research, Vol. 64: 2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules may originate from any mammalian source, such as humans, mice, rats, rabbits, pigs, dogs, cats, horses, donkeys, guinea pigs, goats, or camelids. Antibody fragments can compete with intact antibodies for binding to target antigens, and fragments can be produced by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or newly synthesized using recombinant DNA technology or peptide synthesis. Antigen-binding molecules may include, for example, selective protein scaffolds or artificial scaffolds grafted with CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced to stabilize the three-dimensional structure of the antigen-binding molecule, and, for example, fully synthetic scaffolds containing biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129 (2003), and Roque et al., Biotechnol. Prog. 20:639654 (2004). Furthermore, peptide antibody mimetics ("PAM") and scaffolds based on antibody mimetics that utilize fibronectin components as a scaffold can be used.

[0030] Antigen-binding molecules may also include proteins containing one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen-binding molecules include diabodies (see, e.g., European Patent No. 404,097, International Publication No. 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90:6444-6448, 1993), intrabodies, domain antibodies (a single VL or VH domain, or two or more VH domains linked by a peptide linker, see Ward et al., Nature, Vol. 341:544546, 1989), and maxibodies (two scFv fused to an Fc region, see Fredericks et al., Protein Engineering, Design & Selection, Vol. 17:95-106, 2004 and Powers et al., Journal of Immunological Sciences). See Methods, Vol.251:123-135, 2001), triabodies, tetrabodies, minibodies (scFv fused to the CH3 domain, see Olafsen et al., Protein Eng Des Sel., Vol.17:315-23, 2004), peptidebodies (one or more peptides bound to the Fc region, see International Publication No. 00 / 24782), linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide, see Zapata et al., Protein Examples include, but are not limited to, eng., Vol.8:1057-1062, 1995, miniature modular immunotherapies (see U.S. Patent Application Publication No. 20030133939), and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc). In certain embodiments, the antigen-binding molecule may have, for example, the structure of an immunoglobulin.

[0031] As used herein, the term “antigen-binding fragment” (Fab) has its plain, ordinary meaning when read in light of this specification and refers to the portion of an antibody that binds to an antigen. As used herein, Fab may refer to both monovalent F(ab) and divalent F(ab')2 fragments. An antibody digested by the enzyme papain yields two F(ab) and Fc fragments, each approximately 50 kDa. In contrast, an F(ab')2 fragment antibody can be produced by pepsin digestion of a whole IgG antibody to remove most of the Fc region while leaving a portion of the hinge region intact. An F(ab')2 fragment has two antigen-binding F(ab) moieties linked together by a disulfide bond and is therefore divalent with a molecular weight of approximately 110 kDa.

[0032] The “knob-in-hole” strategy (see, for example, International Publication No. 2006 / 028936) has its plain, ordinary meaning when read in light of this specification and refers to a strategy that can be used to produce full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at a position that affects the CH3 domain interaction to promote heterodimerization. An amino acid with a small side chain (the hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (the knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction between the heavy chain with the “hole” and the heavy chain with the “knob”.

[0033] As used herein, the term “electrostatic steering” has its plain, ordinary meaning as understood in light of this specification, and refers to a mechanism for enhancing the binding rate and affinity of an ionic ligand by a countercharge distribution that induces an induced potential field.

[0034] As used herein, the term “charge-charge pairing” has its plain, ordinary meaning as understood in light of this specification and refers to the electrostatic interaction between two charged particles. These interactions are, in some cases, the strongest non-covalent forces and can extend over longer distances than other non-covalent interactions.

[0035] As used herein, “pharmaceutically acceptable” means carriers, excipients, and / or stabilizers in their plain, ordinary meaning as understood herein, which are non-toxic or have an acceptable level of toxicity to cells or mammals to which they are exposed at the dosages and concentrations used. As used herein, “pharmaceutically acceptable” “diluents,” “excipients,” and / or “carriers” mean any solvent, dispersible medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., which are suitable for administration to human, cat, dog, or other vertebrate hosts. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are diluents, excipients, and / or carriers approved by federal, state, or other regulatory authorities for use in animals, including humans and non-human mammals such as cats and dogs, or listed in the United States Pharmacopeia or other commonly recognized pharmacopoeias. The terms diluent, excipient, and / or carrier may refer to a diluent, adjuvant, excipient, or vehicle administered with a pharmaceutical preparation. Such pharmaceutical diluents, excipients, and / or carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin. Water, physiological saline, and aqueous dextrose and glycerol solutions may be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starches, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, salts, sodium chloride, dried skim milk, glycerin, propylene, glycol, water, and ethanol. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers may include one or more of the following: antioxidants such as ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; carbohydrates such as amino acids, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as glycerol, erythritol, trethitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, isomalt, maltitol, or lactitol; salt-forming counterions such as sodium; and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. Optionally, the formulation may further contain small amounts of wetting agents, bulk agents, emulsifiers, or pH buffers. These formulations may take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should be suitable for the mode of administration.

[0036] Additional excipients with desirable properties include preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), citric acid, ascorbic acid, acetic acid, salts, phosphates, citrates, acetates, succinates, chlorides, bicarbonates, borates, sulfates, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, dextran 40, fructose, mannose, lactose, trehalose, galactose, sucrose, sorbitol, mannitol, and cellulose. Examples of substances that may be used include, but are not limited to, serum, amino acids, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer, poloxamer 188, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be present in residues from the manufacturing process or in contaminants, and include, but are not limited to, serum, albumin, ovalbumin, antibiotics, inactivators, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components, or any combination thereof.The amount of excipients may be found in the formulation as 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, approximately that number, at least that number, at least approximately that number, less than or equal to that number, or approximately less than or equal to that number, or any weight percentage within the range defined by any two of the numbers above.

[0037] The term “purity” used herein for any given substance, compound, or material has its plain, ordinary meaning when read in light of this specification and refers to the actual amount of the substance, compound, or material relative to the expected amount. For example, a substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by undesirable impurities, including but not limited to by-products, isomers, enantiomers, decomposition products, solvents, carriers, vehicles, or contaminants, or any combination thereof. Purity can be measured by techniques including, but not limited to, chromatography, liquid chromatography, gas chromatography, spectroscopy, UV-Vis spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetric or titration, or any combination thereof.

[0038] The term “pharmaceutically acceptable salt” has its plain, ordinary meaning as understood herein and includes relatively non-toxic inorganic and organic acids, or base addition salts, of compositions or excipients, including but not limited to analgesics, therapeutic agents, and other materials. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of inorganic bases suitable for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed using suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, such a class of organic bases may include, but are not limited to, mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids such as glycine, arginine, and lysine; guanidine, N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0039] This Disclosure Embodiment Some embodiments described herein relate to Fab, antibodies, compositions comprising Fab and / or antibodies, and methods for producing Fab and / or antibodies. In some embodiments, Fab, antibodies, or residues within their domains are referred to in relation to the position of residues within Fab, antibodies, or their domains according to one or more numbering rules, e.g., the EU Kabat numbering system. Figure 14 is a table showing some embodiments of CH1 sequence alignment for the wild-type and mutant CH1 domains of IHG1. Figure 15 is a table showing some embodiments of CL sequence alignment for the lambda and mutant CL domains. Figure 16 is a table showing some embodiments of CL sequence alignment for the kappa and mutant CL domains. Unless otherwise specified, all references to specific substitutions in numbered residues utilize the European numbering system.

[0040] This specification discloses embodiments relating to antigen-binding fragments (Fabs).

[0041] Figure 1 is a diagram of an embodiment of an antigen-binding fragment (Fab). In Figure 1, Fab 100 comprises the heavy chain (HC) variable domain (VH) 101, the heavy chain (HC) constant domain (CH1) 102, the light chain (LC) variable domain (VL) 103, and the light chain (LC) constant domain (CL) 104 of human IgG, wherein HC CH1 includes substitutions to F170, S183, and V185 (EU numbering), LC CL includes substitution to L135 (EU numbering), HC1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and CL is This does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, CH1 consists of substitutions in F170, S183, and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, the HC CH1 substitution in F170 (EU numbering) is the F170I or F170V substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is the S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the LC CL substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, HC CH1 includes the F170I, S183L, and V185L (EU numbering) substitutions, and LC CL includes the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of sequence numbers 15, 19, 23, or 24, and LC CL includes or consists of sequence numbers 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of sequence number 9, and LC CL includes substitutions in L29 of sequence number 1 or L28 of sequence number 6.In some embodiments, the HC CH1 substitution at F53 of SEQ ID NO: 9 is F53I or F53V substitution. In some embodiments, the HC CH1 substitution at S66 of SEQ ID NO: 9 is S66L or S66I substitution. In some embodiments, the HC CH1 substitution at V68 of SEQ ID NO: 9 is V68L substitution. In some embodiments, the LC CL substitution at L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is L29F or L28F substitution. In some embodiments, Fab is a chimeric or humanized Fab. In some embodiments, Fab is a human Fab.

[0042] In some embodiments, the antigen-binding fragment (Fab) comprises a human IgG heavy chain (HC) variable domain (VH), a human IgG heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a human IgG light chain (LC) constant domain (CL), where HC CH1 includes substitutions to F126, F170, S183, V185, and C220 (EU numbering), and LC CL includes substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes substitutions to E124, L135, and C214 (lambda), and in some embodiments, LC CL includes substitutions for Q124, L135, and C214 (Kappa), and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, CH1 consists of substitutions in F126, F170, S183, V185, and C220 (EU numbering), and LC CL consists of substitutions in E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering). In some embodiments, LC CL consists of substitutions in E124, L135, and C214 (lambda). In some embodiments, LC CL consists of substitutions in Q124, L135, and C214 (kappa). In some embodiments, HC CH1 substitution in F126 (EU numbering) is F126C substitution. In some embodiments, HC CH1 substitution in F170 (EU numbering) is F170V or F170I substitution. In some embodiments, HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes substitutions for E124C, L135F, and C214S (EU numbering). In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (Kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of SEQ ID NOs. 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs. 2, 5, 7, or 8. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO. 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO. 1.In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of sequence number 9, and LC CL consists of substitutions in Q17, L28, and C107 of sequence number 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution at L29 in SEQ ID NO: 1 is the L29F substitution. In some embodiments, the LC CL1 substitution at C107 in SEQ ID NO: 6 is the C107S substitution. In some embodiments, the LC CL1 substitution at C105 in SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6. In some embodiments, Fab is a chimeric or humanized Fab.In some embodiments, Fab is human Fab.

[0043] In some embodiments, the antigen-binding fragment (Fab) comprises a human IgG heavy chain (HC) variable domain (VH), a human IgG heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a human IgG light chain (LC) constant domain (CL), wherein HC CH1 includes substitutions to F126 and C220 (EU numbering), and LC CL includes substitutions to E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes substitutions to E124 and C214 (lambda), and in some embodiments, LC CL includes substitutions for Q124 and C214 (Kappa), and HC1 is selected from L128F, A141M, A141T, A141I, F170I, F170M, F170Y, F170S, F170A, F170V, S181I, S181T, S181M, S183A, S183L, S183I, S183V, V185L and V185A (EU numbering). CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (European numbering). In some embodiments, the HC CH1 substitution in F126 (EU numbering) is the F126C substitution, and the LC CL substitution in E124 (lambda) or Q124 (kappa) (EU numbering) is the E124C or Q124C substitution. In some embodiments, the LC CL substitution in E124 (lambda) is the E124C substitution. In some embodiments, the LC-CL substitution in Q124 (kappa) is the Q124C substitution. In some embodiments, the HC-CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC-CL substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, Fab is a chimeric or humanized Fab. In some embodiments, Fab is a human Fab.

[0044] In some embodiments, the antigen-binding fragment (Fab) comprises a human IgG heavy chain (HC) variable domain (VH), a human IgG heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a human IgG light chain (LC) constant domain (CL), wherein the LC VL framework region (FR) and / or the HC VH framework region (FR) contain one or more substitutions. In some embodiments, the VL FR contains substitutions for Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, the VH FR contains substitutions for Q39 and / or G44 (EU numbering). In some embodiments, the VH FR contains substitutions for Q39 and / or G44 (EU numbering), and the VL FR contains substitutions for Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, the VL FR substitution in Q38 includes or consists of substitutions selected from the group consisting of Q38D and Q38K substitutions (EU numbering). In some embodiments, the VL FR substitution in G100 includes or consists of substitutions selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering). In some embodiments, the VL FR substitution in G101 includes or consists of substitutions selected from the group consisting of G101D and G101K substitutions (EU numbering). In some embodiments, the VH FR substitution in Q39 includes or consists of substitutions selected from the group consisting of Q39D and Q39K substitutions (EU numbering). In some embodiments, the VH FR substitution in G44 includes or consists of substitutions selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering). In some embodiments, the substitution of VH FR includes or consists of the Q39K substitution, and the substitution of VL FR includes or consists of the Q38D substitution. In some embodiments, the substitution of VH FR includes or consists of the Q39D substitution, and the substitution of VL FR includes or consists of the Q38K substitution. In some embodiments, the substitution of VH FR includes or consists of the G44K substitution, and the substitution of VL FR includes or consists of the G100D substitution.In some embodiments, the substitution of VH FR includes or consists of the G44D substitution, and the substitution of VL FR includes or consists of the G100K substitution. In some embodiments, the substitution of VH FR includes or consists of the G44C substitution, and the substitution of VL FR includes or consists of the G100C substitution. In some embodiments, the substitution of VH FR includes or consists of the G44K substitution, and the substitution of VL FR includes or consists of the G101D substitution. In some embodiments, the substitution of VH FR includes or consists of the G44D substitution, and the substitution of VL FR includes or consists of the G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of the Q39K substitution, the substitution of LC1 VL FR includes or consists of the Q38D substitution, the substitution of HC2 VH FR includes or consists of the Q39D substitution, and the substitution of LC2 VL FR includes or consists of the Q38K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G100D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G100K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44D substitution, the substitution of LC1 VL FR includes or consists of G100K substitution, the substitution of HC2 VH FR includes or consists of G44K substitution, and the substitution of LC2 VL FR includes or consists of G100D substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution.In some embodiments, the substitution of HC2 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G101D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44D substitution, the substitution of LC1 VL FR includes or consists of G101K substitution, the substitution of HC2 VH FR includes or consists of G44K substitution, and the substitution of LC2 VL FR includes or consists of G101D substitution.

[0045] Antibodies are disclosed herein. In some embodiments, the antibody comprises one or more Fab fragments, as illustrated in Figure 1 and described above elsewhere herein. Figure 2 is a diagram showing embodiments of the antibody. Some embodiments herein relate to an antibody 200 or fragment thereof comprising a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, respectively, where each HC1 and HC2 comprises a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a human IgG light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 includes or consists of substitutions with F170 and S183, optionally substitution with V185 (EU numbering), and LC1 CL includes or consists of substitution with L135 (EU numbering). CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the antibody is human IgG. In some embodiments, the antibody is human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, CH1 of HC1 consists of substitutions in F170, S183, and V185 (EU numbering), and CL of LC1 consists of substitutions in L135 (EU numbering).In some embodiments, HC2 CH1 includes or consists of sequence number 9, and LC2 CL includes or consists of sequence number 1 or 6. In some embodiments, the substitution of HC1 CH1 in F170 (EU numbering) is the substitution of F170I or F170V. In some embodiments, the substitution of HC1 CH1 in S183 (EU numbering) is the substitution of S183L or S183I. In some embodiments, the substitution of HC1 CH1 in V185 (EU numbering) is the substitution of V185L. In some embodiments, the substitution of LC1 CL1 in L135 (EU numbering) is the substitution of L135F. In some embodiments, HC1 CH1 includes or consists of the substitutions of F170V, S183I and V185L (EU numbering), and LC1 CL includes or consists of the substitution of L135F (EU numbering). In some embodiments, HC1 CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and LC1 CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of SEQ ID NO: 9, and LC CL includes substitutions in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO: 9 is the F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is the V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is the L29F or L28F substitution.

[0046] In some embodiments, the antibody or its fragment comprises a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1), each LC1 and LC2 comprising the human IgG light chain variable domain (VL) and the light chain constant domain (CL), HC1 CH1 comprising or consisting of substitutions to F126, F170, S183, V185, and C220 (EU numbering), and LC1 CL comprising or consisting of substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes or consists of substitutions for E124, L135, and C214 (lambda), and in some embodiments, LC CL includes or consists of substitutions for Q124, L135, and C214 (kappa), HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the antibody is human IgG. In some embodiments, the antibody is human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the HC CH1 substitution in F126 (EU numbering) is the F126C substitution.In some embodiments, the HC CH1 substitution in F170 (EU numbering) is F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is C220S substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of the F126C, F170V, S183I, V185L, and C220S (EU numbering) substitutions, and LC CL includes the E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering) substitutions. In some embodiments, LC CL includes the E124C, L135F, and C214S (lambda) (EU numbering) substitutions. In some embodiments, LC CL includes the Q124C, L135F, and C214S (kappa) (EU numbering) substitutions. In some embodiments, HC CH1 includes or consists of substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes substitutions E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes substitutions E124C, L135F, and C214S (lambda) (EU numbering).In some embodiments, LC CL includes substitutions of Q124C, L135F, and C214S (Kappa) (EU numbering). In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of SEQ ID NO: 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of SEQ ID NO: 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of SEQ ID NO: 6 is the L28F substitution. In some embodiments, the LC CL1 substitution in L29 of SEQ ID NO: 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of SEQ ID NO: 6 is the C107S substitution. In some embodiments, the LC CL1 substitution in C105 of SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes the Q17C, L28F, and C107S (AA SEQ ID NO number) substitutions. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and E18, L29, and C105 of SEQ ID NO: 1.In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6.

[0047] In some embodiments, the antibody or its fragment comprises a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), and the LC1 and / or LC2 VL framework region (FR) and / or HC1 and / or HC2 VH framework region (FR) comprising one or more substitutions. In some embodiments, the LC1 and / or LC2 VL FR comprises substitutions in Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, the HC1 and / or HC2 VH FR comprises substitutions in Q39 and / or G44 (EU numbering). In some embodiments, HC1 and / or HC2 VH FR include substitutions in Q39 and / or G44 (EU numbering), and LC1 and / or LC2 VL FR include substitutions in Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, the LC1 and / or LC2 VL FR substitution in Q38 includes or consists of substitutions selected from the group consisting of Q38D and Q38K substitutions (EU numbering). In some embodiments, the LC1 and / or LC2 VL FR substitution in G100 includes or consists of substitutions selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering). In some embodiments, the LC1 and / or LC2 VL FR substitution in G101 includes or consists of substitutions selected from the group consisting of G101D and G101K substitutions (EU numbering). In some embodiments, the HC1 and / or HC2 VH FR substitution in Q39 includes or consists of substitutions selected from the group consisting of Q39D and Q39K substitutions (EU numbering). In some embodiments, the HC1 and / or HC2 VH FR substitution in G44 includes or consists of substitutions selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering).In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of Q39K substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of Q38D substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of Q39D substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of Q38K substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of G44K substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of G100D substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of G44D substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of G100K substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of G44C substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of G44K substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of G101D substitution. In some embodiments, the substitution of HC1 and / or HC2 VH FR includes or consists of G44D substitution, and the substitution of LC1 and / or LC2 VL FR includes or consists of G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of the Q39K substitution, the substitution of LC1 VL FR includes or consists of the Q38D substitution, the substitution of HC2 VH FR includes or consists of the Q39D substitution, and the substitution of LC2 VL FR includes or consists of the Q38K substitution.In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G100D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G100K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44D substitution, the substitution of LC1 VL FR includes or consists of G100K substitution, the substitution of HC2 VH FR includes or consists of G44K substitution, and the substitution of LC2 VL FR includes or consists of G100D substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC2 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G101D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44D substitution, the substitution of LC1 VL FR includes or consists of G101K substitution, the substitution of HC2 VH FR includes or consists of G44K substitution, and the substitution of LC2 VL FR includes or consists of G101D substitution.

[0048] In some embodiments, the antibody or its fragment comprises a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising the human IgG heavy chain variable domain (VH) and the first heavy chain constant domain (CH1), each LC1 and LC2 comprising the human IgG light chain variable domain (VL) and the light chain constant domain (CL), and the LC1 and / or LC2 VL framework region (FR) and / or HC1 and / or HC2 VH framework region (FR) comprising one or more substitutions. In some embodiments, HC1 or HC2 VH comprises or consists of one of sequence numbers 53 to 64. In some embodiments, LC1 or LC2 VL comprises or consists of one of sequence numbers 65 to 80. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 30, 43, 35, and 52, or SEQ ID NOs. 35, 52, 30, and 43, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 29, 44, 36, and 51, or SEQ ID NOs. 36, 51, 29, and 44, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 28, 39, 33, and 48, or SEQ ID NOs. 33, 48, 28, and 39, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 27, 40, 34, and 47, or SEQ ID NOs. 34, 47, 27, and 40, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 26, 38, 31, and 45, or SEQ ID NOs. 31, 45, 26, and 38, respectively.In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 25, 37, 32, and 46, or SEQ ID NOs. 32, 46, 25, and 37, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 28, 41, 33, and 50, or SEQ ID NOs. 33, 50, 28, and 41, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 27, 42, 34, and 49, or SEQ ID NOs. 34, 49, 27, and 42, respectively. In some embodiments, HC1 includes or consists of sequence number 30, LC1 includes or consists of sequence number 43, HC2 includes or consists of sequence number 35, and LC2 includes or consists of sequence number 52. In some embodiments, HC1 includes or consists of sequence number 29, LC1 includes or consists of sequence number 44, HC2 includes or consists of sequence number 36, and LC2 includes or consists of sequence number 51. In some embodiments, HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 48. In some embodiments, HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 47. In some embodiments, HC1 includes or consists of sequence number 26, LC1 includes or consists of sequence number 38, HC2 includes or consists of sequence number 31, and LC2 includes or consists of sequence number 45. In some embodiments, HC1 includes or consists of sequence number 25, LC1 includes or consists of sequence number 37, HC2 includes or consists of sequence number 32, and LC2 includes or consists of sequence number 46.In some embodiments, HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 50. In some embodiments, HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 49. In some embodiments, HC1 includes or consists of sequence number 35, LC1 includes or consists of sequence number 52, HC2 includes or consists of sequence number 30, and LC2 includes or consists of sequence number 43. In some embodiments, HC1 includes or consists of sequence number 36, LC1 includes or consists of sequence number 51, HC2 includes or consists of sequence number 29, and LC2 includes or consists of sequence number 44. In some embodiments, HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 48, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 39. In some embodiments, HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 47, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 40. In some embodiments, HC1 includes or consists of sequence number 31, LC1 includes or consists of sequence number 45, HC2 includes or consists of sequence number 26, and LC2 includes or consists of sequence number 38. In some embodiments, HC1 includes or consists of sequence number 32, LC1 includes or consists of sequence number 46, HC2 includes or consists of sequence number 25, and LC2 includes or consists of sequence number 37. In some embodiments, HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 50, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 41.In some embodiments, HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 49, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 42.

[0049] In some embodiments, the antibody or fragment thereof disclosed herein comprises two heavy / light chain pairs, the first pair being HC1 / LC1 and the second pair being HC2 / LC2. In some embodiments, the antibody or fragment thereof disclosed herein further comprises the HC1 and HC2 pair. In some embodiments, HC1 and HC2 are paired via charge-charge interactions. In some embodiments, each HC1 and HC2 further comprises a heavy chain constant domain (CH3), and the CH3 domain of HC1 and the CH3 domain of HC2 together constitute a knob-into-hole construct. In some embodiments, the HC1 CH3 constitutes the knob and the HC2 CH3 constitutes the hole. In some embodiments, the HC1 CH3 constitutes the hole and the HC2 CH3 constitutes the knob. In some embodiments, LC1 and / or LC2 are kappa light chains. In some embodiments, LC1 and / or LC2 are lambda light chains. In some embodiments, HC CH1 includes or consists of SEQ ID NOs: 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs: 2, 5, 7, or 8.

[0050] In some embodiments, the antibodies or fragments thereof disclosed herein comprise a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), wherein HC1 CH1 comprises or consists of substitutions at F126 and C220 (EU numbering), LC1 CL comprises or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 comprises or consists of substitutions at F170, S183, and V185 (EU numbering), and LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), LC1 CL and / or LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the antibody is human IgG. In some embodiments, the antibody is human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the HC1 CH1 substitution in F126 (EU numbering) is the F126C substitution, and the LC1 CL substitution in position 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL substitution in E124 (lambda) is the E124C substitution.In some embodiments, the LC CL substitution in Q124 (Kappa) is the Q124C substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of sequence number 9, and LC CL consists of substitutions in E18, L29, and C105 of sequence number 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of sequence number 9, and LC CL consists of substitutions in Q17, L28, and C107 of sequence number 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is V68L substitution. In some embodiments, the HC CH1 substitution in C103 is C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is L28F substitution. In some embodiments, the LC CL1 substitution in L29 of sequence number 1 is L29F substitution. In some embodiments, the LC CL1 substitution in C107 of sequence number 6 is C107S substitution. In some embodiments, the LC CL1 substitution in C105 of sequence number 1 is C105S substitution. In some embodiments, HC CH1 includes or consists of substitutions for F9C, F53V, S66I, V68L, and C103S, and LC CL includes substitutions for Q17C, L28F, and C107S.In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and Q17, L28, and C107 of SEQ ID NO: 6.

[0051] Some embodiments herein relate to compositions comprising one or more polynucleotides encoding a Fab and / or antibody, comprising a heavy chain (HC) variable domain (VH) 101, a heavy chain (HC) constant domain (CH1) 102, a light chain (LC) variable domain (VL) 103, and a light chain (LC) constant domain (CL) 104 of human IgG, wherein HC CH1 comprises or consists of substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC CL includes or consists of the substitution (EU numbering) for L135, and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the polynucleotide encodes human IgG. In some embodiments, the polynucleotide encodes human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the polynucleotide encodes a chimeric antibody. In some embodiments, the polynucleotide encodes a humanized antibody. In some embodiments, the polynucleotide encodes a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, V185 of CH1 is substituted. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is F170I or F170V substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.In some embodiments, the substitution of HC CH1 in V185 (EU numbering) is the substitution of V185L. In some embodiments, the substitution of LC CL1 in L135 (EU numbering) is the substitution of L135F. In some embodiments, HC CH1 includes or consists of the substitutions F170V, S183I, and V185L (EU numbering), and LC CL includes or consists of the substitution L135F (EU numbering). In some embodiments, HC CH1 includes or consists of the substitutions F170I, S183L, and V185L (EU numbering), and LC CL includes or consists of the substitution L135F (EU numbering). In some embodiments, HC CH1 includes or consists of sequence numbers 15, 19, 23, or 24, and LC CL includes or consists of sequence numbers 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of SEQ ID NO: 9, and LC CL includes substitutions in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO: 9 is F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is L29F or L28F substitution.

[0052] Some embodiments herein relate to compositions comprising one or more polynucleotides encoding a Fab and / or antibody comprising a human IgG heavy chain (HC) variable domain (VH), a heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a light chain (LC) constant domain (CL), wherein HC CH1 includes or consists of substitutions to F126, F170, S183, V185, and C220 (EU numbering), and LC CL includes or consists of substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes or consists of substitutions to E124, L135, and C214 (lambda), and in some embodiments, LC CL includes or consists of substitutions for Q124, L135, and C214 (Kappa), and HC1 is any of the following selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Without substitutions, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the composition contains human IgG. In some embodiments, the composition contains human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the composition contains chimeric antibody. In some embodiments, the composition contains humanized antibody. In some embodiments, the composition contains chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, CH1 includes or consists of substitutions for F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL includes or consists of substitutions for L135 (EU numbering). In some embodiments, the HC CH1 substitution in F126 (EU numbering) is the F126C substitution.In some embodiments, the HC CH1 substitution in F170 (EU numbering) is F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is C220S substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of the F126C, F170V, S183I, V185L, and C220S (EU numbering) substitutions, and LC CL includes or consists of the E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering) substitutions. In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering).In some embodiments, HC CH1 includes or consists of SEQ ID NOs: 15, 19, 123, or 24, and LC CL includes or consists of SEQ ID NOs: 2, 5, 7, or 8. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution in L29 of sequence number 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of sequence number 6 is the C107S substitution. In some embodiments, the LC CL1 substitution in C105 of sequence number 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1.In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6.

[0053] In some embodiments, the compositions disclosed herein comprise a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), wherein HC1 CH1 comprises or consists of substitutions at F126 and C220 (EU numbering), LC1 CL comprises or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 comprises or consists of substitutions at F170, S183, and V185 (EU numbering), and LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), LC1 CL and / or LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the composition contains human IgG. In some embodiments, the composition contains human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the composition contains a chimeric antibody. In some embodiments, the composition contains a humanized antibody. In some embodiments, the composition contains a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the HC1 CH1 substitution in F126 (EU numbering) is the F126C substitution, and the LC1 CL substitution in position 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) substitution.In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the HC CH1 substitution at C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL substitution at C214 (EU numbering) is the C214S substitution.

[0054] Some embodiments herein relate to expression vectors or constructs for the expression of one or more polynucleotides encoding a Fab and / or antibody comprising a human IgG heavy chain (HC) variable domain (VH) 101, a heavy chain (HC) constant domain (CH1) 102, a human IgG light chain (LC) variable domain (VL) 103, and a light chain (LC) constant domain (CL) 104, wherein HC CH1 comprises or consists of substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC CL includes or consists of the substitution (EU numbering) for L135, and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183, and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, V185 of CH1 is substituted. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is the F170I or F170V substitution. In some embodiments, the expression vector or construct expresses a human antibody or a fragment thereof. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the expression vector or construct expresses human IgG. In some embodiments, the expression vector or construct expresses human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the expression vector or construct expresses a chimeric antibody. In some embodiments, the expression vector or construct expresses a humanized antibody.In some embodiments, the expression vector or construct expresses a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is a V185L substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is an L135F substitution. In some embodiments, HC CH1 includes or consists of F170I and S183V (EU numbering) substitutions, and LC CL includes or consists of an L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of F170I, S183L, and V185L (EU numbering) substitutions, and LC CL includes an L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of SEQ ID NOs: 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs: 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of SEQ ID NO: 9, and LC CL includes substitutions in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO: 9 is the F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is the V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is the L29F or L28F substitution.

[0055] Some embodiments herein relate to expression vectors or constructs for the expression of one or more polynucleotides encoding a Fab and / or antibody comprising a heavy chain (HC) variable domain (VH), a heavy chain (HC) constant domain (CH1), a light chain (LC) variable domain (VL), and a light chain (LC) constant domain (CL) of human IgG, wherein HC CH1 includes or consists of substitutions to F126, F170, S185, V185, and C220 (EU numbering), and LC CL includes or consists of substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes or consists of substitutions to E124, L135, and C214 (lambda), and in some embodiments, LC CL includes or consists of substitutions for Q124, L135, and C214 (Kappa), and HC1 is any of the following selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Excluding substitutions, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the expression vector or construct expresses a polynucleotide encoding a human antibody or a fragment thereof. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a humanized antibody.In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, CH1 contains or consists of substitutions in F170 and S183 (EU numbering), or F170, S183, and V185 (EU numbering), and CL contains or consists of substitutions in L135 (EU numbering). In some embodiments, the HC CH1 substitution in F126 (EU numbering) is an F126C substitution. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is an F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is a V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of substitutions F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering).In some embodiments, HC CH1 includes or consists of substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes substitutions E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes substitutions E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes substitutions Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of sequence numbers 15, 19, 23, or 24, and LC CL includes or consists of sequence numbers 2, 5, 7, or 8. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution in L29 of sequence number 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of sequence number 6 is the C107S substitution.In some embodiments, the LC CL1 substitution at C105 in SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes the Q17C, L28F, and C107S substitutions. In some embodiments, HC CH1 includes or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes the E18C, L29F, and C105S substitutions in positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6.

[0056] Some embodiments herein relate to expression vectors or constructs for the expression of one or more polynucleotides encoding a Fab and / or antibody comprising a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, where each HC1 and HC2 comprises a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a human IgG light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 includes or consists of substitutions at F126 and C220 (EU numbering), LC1 CL includes or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 includes or consists of substitutions at F170, S183, and V185 (EU numbering), and LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V and V185A (EU numbering), LC1 CL and / or LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the expression vector or construct expresses a polynucleotide encoding a human antibody or a fragment thereof. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric antibody.In some embodiments, the expression vector or construct expresses a polynucleotide encoding a humanized antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the HC1 CH1 substitution at F126 (EU numbering) is an F126C substitution, and the LC1 CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is an E124C (lambda) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is a Q124C (kappa) substitution. In some embodiments, the HC CH1 substitution at C220 (EU numbering) is a C220S substitution. In some embodiments, the LC CL substitution at C214 (EU numbering) is a C214S substitution.

[0057] Some embodiments herein relate to cells comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody, including a heavy chain (HC) variable domain (VH) 101, a heavy chain (HC) constant domain (CH1) 102, a light chain (LC) variable domain (VL) 103, and a light chain (LC) constant domain (CL) 104 of human IgG, wherein HC CH1 includes or comprises substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC CL includes or consists of the substitution (EU numbering) for L135, and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183, and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, the HC CH1 substitution in F170 (EU numbering) is the F170I or F170V substitution. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a chimeric antibody.In some embodiments, the cell comprises one or more polynucleotides encoding a humanized antibody, an expression vector, or a construct. In some embodiments, the cell comprises one or more polynucleotides encoding a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody, an expression vector, or a construct. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is a V185L substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is an L135F substitution. In some embodiments, HC CH1 comprises or consists of F170I and S183V (EU numbering) substitutions, and LC CL comprises or consists of an L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and LC CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of SEQ ID NOs. 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs. 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions for F53, S66, and V68 of SEQ ID NO. 9, and LC CL includes substitutions for L29 of SEQ ID NO. 1 or L28 of SEQ ID NO. 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO. 9 is the F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO. 9 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO. 9 is the V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is an L29F or L28F substitution. In some embodiments, the cells are mammalian cells or bacterial cells. In some embodiments, the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells.

[0058] Some embodiments herein relate to cells comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody, including a human IgG heavy chain (HC) variable domain (VH), a heavy chain (HC) constant domain (CH1), a human IgG light chain (LC) variable domain (VL), and a light chain (LC) constant domain (CL), wherein HC CH1 includes or consists of substitutions with F126, F176, S189, V191, and C220 (EU numbering), and LC CL includes or consists of substitutions for E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and HC1 includes L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). CL does not include any substitutions selected from (EU numbering), and does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of SEQ ID NO: 9, and LC CL includes substitutions in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO: 9 is F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is L29F or L28F substitution.In some embodiments, the cell comprises one or more polynucleotides encoding a human antibody or a fragment thereof, an expression vector, or a construct. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides encoding human IgG, an expression vector, or a construct. In some embodiments, the cell comprises one or more polynucleotides encoding human IgG1, IgG2, IgG3, or IgG4 antibodies, an expression vector, or a construct. In some embodiments, the cell comprises one or more polynucleotides encoding a chimeric antibody, an expression vector, or a construct. In some embodiments, the cell comprises one or more polynucleotides encoding a polynucleotide encoding a humanized antibody, an expression vector, or a construct. In some embodiments, the HC CH1 substitution in F126 (EU numbering) is an F126C substitution. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is an F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution.In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of SEQ ID NOs: 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs: 2, 5, 7, or 8. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution.In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution in L29 of sequence number 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of sequence number 6 is the C107S substitution. In some embodiments, the LC CL1 substitution in C105 of sequence number 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S (EU numbering). In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes Q17C, L28F, and C107S substitutions in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6. In some embodiments, the cells are mammalian cells or bacterial cells. In some embodiments, the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells.

[0059] Some embodiments herein relate to cells comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody comprising a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, wherein each HC1 and HC2 comprises a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a human IgG light chain variable domain (VL) and a light chain constant domain (CL), wherein HC1 CH1 contains or consists of substitutions at F126 and C220 (EU numbering), LC1 CL contains or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 contains or consists of substitutions at F170, S183, and V185 (EU numbering), and LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V and V185A (EU numbering), LC1 CL and / or LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the cells include one or more polynucleotides, expression vectors, or constructs encoding chimeric antibodies.In some embodiments, the cell comprises one or more polynucleotides encoding a humanized antibody, an expression vector, or a construct. In some embodiments, the HC1 CH1 substitution at F126 (EU numbering) is an F126C substitution, and the LC1 CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is an E124C (lambda) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is a Q124C (kappa) substitution. In some embodiments, the HC CH1 substitution at C220 (EU numbering) is a C220S substitution. In some embodiments, the LC CL substitution at C214 (EU numbering) is a C214S substitution. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution.

[0060] In some embodiments, the LC CL1 substitution in L29 of SEQ ID NO: 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of SEQ ID NO: 6 is the C107S substitution. In some embodiments, the LC CL1 substitution in C105 of SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes the Q17C, L28F, and C107S (EU numbering) substitutions. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in positions corresponding to F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and Q17, L28, and C107 of SEQ ID NO: 6.

[0061] Some embodiments herein relate to pharmaceutical compositions comprising a Fab and / or antibody comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody, comprising a heavy chain (HC) variable domain (VH) 101, a heavy chain (HC) constant domain (CH1) 102, a light chain (LC) variable domain (VL) 103, and a light chain (LC) constant domain (CL) 104 of human IgG, wherein HC CH1 comprises or consists of substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC CL includes or consists of the substitution (EU numbering) for L135, and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the pharmaceutical composition comprises a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises human IgG. In some embodiments, the pharmaceutical composition comprises IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the pharmaceutical composition comprises a chimeric antibody. In some embodiments, the pharmaceutical composition comprises a polynucleotide encoding a humanized antibody. In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, V185 of CH1 is substituted. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is F170I or F170V substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, HC CH1 includes or consists of the F170I and S183V (EU numbering) substitutions, and LC CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and LC CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of sequence numbers 15, 19, 23, or 24, and LC CL includes or consists of sequence numbers 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of SEQ ID NO: 9, and LC CL includes substitutions in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution in F53 of SEQ ID NO: 9 is an F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is an S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is a V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is an L29F or L28F substitution. In some embodiments, the pharmaceutical composition is a cell culture.

[0062] Some embodiments herein are directed to pharmaceutical compositions comprising one or more polynucleotides, expression vectors, or constructs encoding Fabs and / or antibodies comprising the heavy chain (HC) variable domain (VH), heavy chain (HC) constant domain (CH1), light chain (LC) variable domain (VL), and light chain (LC) constant domain (CL) of human IgG, wherein HC CH1 comprises or consists of substitutions at F126, F176, S189, V191, and C220 (EU numbering), and LC CL comprises or consists of substitutions at E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL comprises or consists of substitutions at E124, L135, and C214 (lambda); in some embodiments, LC CL comprises or consists of substitutions at Q124, L135, and C214 (kappa)), HC1 does not comprise any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and CL does not comprise any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the pharmaceutical composition comprises a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises human IgG. In some embodiments, the pharmaceutical composition comprises an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the pharmaceutical composition comprises a chimeric antibody. In some embodiments, the pharmaceutical composition comprises a polynucleotide encoding a humanized antibody. In some embodiments, CH1 consists of substitutions at F170 and S183 (EU numbering), or at F170, S183, and V185 (EU numbering), and CL consists of substitutions at L135 (EU numbering).In some embodiments, the HC CH1 substitution in F126 (EU numbering) is the F126C substitution. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is the F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S189 (EU numbering) is the S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution at position L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution at position C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of the F126C, F170V, S183I, V185L, and C220S (EU numbering) substitutions, and LC CL includes or consists of the E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering) substitutions. In some embodiments, LC CL includes or consists of the E124C, L135F, and C214S (lambda) (EU numbering) substitutions. In some embodiments, LC CL includes or consists of the substitutions Q124C, L135F, and C214S (Kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of the substitutions E124C, L135F, and C214S (Lambda) or Q124C, L135F, and C214S (Kappa) (EU numbering).In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of SEQ ID NOs. 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs. 2, 5, 7, or 8. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO. 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO. 1. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of sequence number 9, and LC CL consists of substitutions in Q17, L28, and C107 of sequence number 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution in L29 of SEQ ID NO: 1 is the L29F substitution. In some embodiments, the LC CL1 substitution in C107 of SEQ ID NO: 6 is the C107S substitution. In some embodiments, the LC CL1 substitution in C105 of SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL includes the Q17C, L28F, and C107S (EU numbering) substitutions.In some embodiments, HC CH1 contains or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL contains the E18C, L29F, and C105S substitutions in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1. In some embodiments, HC CH1 contains or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL contains the Q17C, L28F, and C107S substitutions in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6. In some embodiments, the pharmaceutical composition is a cell culture.

[0063] In some embodiments, the compositions disclosed herein include cell supernatant and / or cell lysates of cells comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody, comprising human IgG heavy chain (HC) variable domain (VH) 101, heavy chain (HC) constant domain (CH1) 102, human IgG light chain (LC) variable domain (VL) 103, and light chain (LC) constant domain (CL) 104, wherein HC CH1 comprises or consists of substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC CL includes or consists of the substitution (EU numbering) for L135, and HC1 includes any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Furthermore, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V and T178I (EU numbering). In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the cell comprises one or more polynucleotides encoding a chimeric antibody, an expression vector, or a construct. In some embodiments, the cell comprises one or more polynucleotides encoding a humanized antibody, an expression vector, or a construct.In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, V185 of CH1 is substituted. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is F170I or F170V substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is V185L substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is L135F substitution. In some embodiments, HC CH1 includes or consists of the F170I and S183V (EU numbering) substitutions, and LC CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and LC CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes or consists of sequence numbers 15, 19, 23, or 24, and LC CL includes or consists of sequence numbers 2, 5, 7, or 8. In some embodiments, HC CH1 includes substitutions for F53, S66, and V68 of sequence number 9, and LC CL includes substitutions for L29 of sequence number 1 or L28 of sequence number 6. In some embodiments, the HC CH1 substitution in F53 of sequence number 9 is the F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is an S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is a V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is an L29F or L28F substitution. In some embodiments, the pharmaceutical composition is a cell culture.

[0064] In some embodiments, the compositions disclosed herein include cell supernatant and / or cell lysates of cells comprising one or more polynucleotides, expression vectors, or constructs encoding a Fab and / or antibody comprising a heavy chain (HC) variable domain (VH), a heavy chain (HC) constant domain (CH1), a light chain (LC) variable domain (VL), and a light chain (LC) constant domain (CL) of human IgG, wherein HC CH1 comprises or consists of substitutions to F126, F176, S189, V191, and C220 (EU numbering), and LC CL comprises or consists of substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL comprises or consists of substitutions to E124, L135, and C214 (lambda), and in some embodiments, LC CL includes or consists of substitutions for Q124, L135, and C214 (Kappa), and HC1 is any of the following selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). Without substitutions, CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a human antibody or a fragment thereof. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the cells include one or more polynucleotides, expression vectors, or constructs encoding chimeric antibodies.In some embodiments, the cell comprises one or more polynucleotides encoding a humanized antibody, an expression vector, or a construct. In some embodiments, CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering). In some embodiments, the HC CH1 substitution in F126 (EU numbering) is an F126C substitution. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is an F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is a V185L substitution. In some embodiments, the HC CH1 substitution in C220 (EU numbering) is a C220S substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is E124C (lambda) or Q124C (kappa). In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is E124C (lambda). In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is Q124C (kappa). In some embodiments, the LC CL1 substitution at position 135 (EU numbering) is L135F. In some embodiments, the LC CL1 substitution at position C214 (EU numbering) is C214S.

[0065] In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes or consists of SEQ ID NOs: 15, 19, 23, or 24, and LC CL includes or consists of SEQ ID NOs: 2, 5, 7, or 8. In some embodiments, the pharmaceutical composition is a cell culture. In some embodiments, CH1 consists of substitutions at F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions at E18, L29, and C105 of SEQ ID NO: 1. In some embodiments, CH1 consists of substitutions at F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions at Q17, L28, and C107 of SEQ ID NO: 6. In some embodiments, the HC CH1 substitution at F9 is an F9C substitution. In some embodiments, the HC CH1 substitution at F53 is an F53V or F53I substitution.In some embodiments, the HC CH1 substitution in S66 is S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is V68L substitution. In some embodiments, the HC CH1 substitution in C103 is C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is L28F substitution. In some embodiments, the LC CL1 substitution in L29 of sequence number 1 is L29F substitution. In some embodiments, the LC CL1 substitution in C107 of sequence number 6 is C107S substitution. In some embodiments, the LC CL1 substitution in C105 of sequence number 1 is C105S substitution. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions E18C, L29F, and C105S in positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6.

[0066] In some embodiments, the compositions disclosed herein include an antibody comprising two heavy / light chain pairs selected from the pairs HC1 / LC1, HC2 / LC2, HC1 / LC2, and HC2 / LC1, wherein at least, or at least about, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibody comprises either an HC1 / LC1 pair and an HC2 / LC2 pair, or a percentage of HC1 / LC1 pairs and HC2 / LC2 pairs within the range defined by any two of the aforementioned values. For example, in some embodiments, the composition comprises an antibody containing two heavy / light chain pairs selected from the pairs HC1 / LC1, HC2 / LC2, HC1 / LC2, and HC2 / LC1, with antibody concentrations of approximately 50-100, 50-95, 50-90, 50-80, 50-75, 50-70, 50-60, 50-55, 55-100, 55-95, 55-90, 55-80, and 55-75. , 55-70, 55-60, 60-100, 60-95, 60-90, 60-80, 60-75, 60-70, 70-100, 70-95, 70-90, 70-80, 75-100, 75-95, 75-90, 75-80, 80-100, 80-95, 80-90, 90-100, 90-95, or 95-100% contain HC1 / LC1 pairs and HC2 / LC2 pairs.

[0067] In some embodiments, the compositions disclosed herein either do not contain an antibody comprising either HC1 / LC2 or HC2 / LC1, or contain an antibody comprising 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less, or contain a percentage of HC1 / LC2 pairs or HC2 / LC1 pairs within the range defined by any two of the aforementioned values. For example, in some embodiments, the compositions disclosed herein include 0.1-50, 0.1-40, 0.1-20, 0.1-25, 0.1-20, 0.1-10, 0.1-5, 0.1-1, 1-50, 1-40, 1-30, 1-25, 1-20, 1-20, 1-5, 5-50, 5-40, 5-30, 5-25, 5-10, 10-50, 10-40, 10-25, 25-50, 25-40, 25-30, 30-50, 30-40, or 40-50% of an HC1 / LC2 pair or an HC2 / LC1 pair.

[0068] In some embodiments, the compositions disclosed herein are cell culture supernatants and / or cell lysates, the compositions containing at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibody containing the HC1 / LC1 pair and the HC2 / LC2 pair, or containing percentages of the HC1 / LC1 pair and the HC2 / LC2 pair within the range defined by any two of the aforementioned values, before any concentration of the HC1 / LC1 pair and / or the HC2 / LC2 pair. For example, in some embodiments, the compositions disclosed herein are enriched to approximately 50-100, 50-95, 50-90, 50-80, 50-75, 50-70, 50-60, 50-55, 55-100, 55-95, 55-90, 55-80, 55-75, 55-70, 55-60 before any enrichment of the HC1 / LC1 pair and / or HC2 / LC2 pair. , containing HC1 / LC1 pairs and / or HC2 / LC2 pairs in the following proportions: 60-100, 60-95, 60-90, 60-80, 60-75, 60-70, 70-100, 70-95, 70-90, 70-80, 75-100, 75-95, 75-90, 75-80, 80-100, 80-95, 80-90, 90-100, 90-95, or 95-100%.

[0069] In some embodiments disclosed herein, the composition contains no antibody or fragment thereof containing either an HC1 / LC2 pair or an HC2 / LC1 pair, or contains 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less, or contains a percentage of an HC1 / LC2 pair or an HC2 / LC1 pair within the range defined by any two of the aforementioned values, before any concentration of the composition for an antibody containing an HC1 / LC1 pair and an HC2 / LC2 pair. For example, in some embodiments, the composition contains about 0.1-50, 0.1-40, 0.1-20, 0.1-25, 0.1-20, 0.1-10, 0.1-5, 0.1-1, 1-50, 1-40, 1-30, 1-25, 1-20, 1-20, 1-5, 5-50, 5-40, 5-30, 5-25, 5-10, 10-50, 10-40, 10-25, 25-50, 25-40, 25-30, 30-50, 30-40, or 40-50% of an HC1 / LC2 pair or an HC2 / LC1 pair before any enrichment of an HC1 / LC1 pair and / or an HC2 / LC2 pair. In some embodiments, the fragment contains an antigen-binding fragment.

[0070] This specification discloses methods for producing antibodies or fragments thereof. Figure 3 is a flowchart showing some embodiments of the methods for producing antibodies or fragments thereof. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the method for producing antibody 300 or fragments thereof includes providing a first heavy chain (HC1) 301, a second heavy chain (HC2) 302, a first light chain (LC1) 303, and a second (LC2) light chain 304, where each HC1 and HC2 comprises a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprises a human IgG light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 includes or consists of substitutions with F170 and S183, and optionally substitution with V185 (EU numbering), and LC1 CL includes or consists of substitution with L135 (EU numbering), and HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, HC1 CH1 includes or consists of substitutions in F170 and S183 (EU numbering), or F170, S183, and V185 (EU numbering), and LC1 CL includes or consists of substitutions in L135 (EU numbering). In some embodiments, HC1 CH1 includes substitutions in V185. In some embodiments, the HC1 CH1 substitution in F170 (EU numbering) is F170I or F170V substitution. In some embodiments, the HC1 CH1 substitution in S183 (EU numbering) is S183L or S183I substitution. In some embodiments, the HC1 CH1 substitution in V185 (EU numbering) is V185L substitution.In some embodiments, the LC1 CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, HC1 CH1 includes or consists of the F170I and S183V (EU numbering) substitutions, and LC1 CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC1 CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and LC1 CL includes or consists of the L135F (EU numbering) substitution. In some embodiments, HC CH1 includes substitutions in F53, S66, and V68 of sequence number 9, and LC CL includes substitutions in L29 of sequence number 1 or L28 of sequence number 6. In some embodiments, the HC CH1 substitution in F53 of sequence number 9 is the F53I or F53V substitution. In some embodiments, the HC CH1 substitution in S66 of SEQ ID NO: 9 is an S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 of SEQ ID NO: 9 is a V68L substitution. In some embodiments, the LC CL substitution in L29 of SEQ ID NO: 1 or L28 of SEQ ID NO: 6 is an L29F or L28F substitution. In some embodiments, the antibody is human IgG. In some embodiments, the antibody is human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody.

[0071] In some embodiments, a method for producing an antibody or its fragment comprises providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), where HC1 CH1 includes or consists of substitutions to F126, F170, S183, V185, and C220 (EU numbering), and LC1 CL includes or consists of substitutions to E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering) (in some embodiments, LC CL includes or consists of substitutions for E124, L135, and C214 (lambda), and in some embodiments, LC CL includes or consists of substitutions for Q124, L135, and C214 (kappa), HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the HC CH1 substitution in F126 (EU numbering) is the F126C substitution. In some embodiments, the HC CH1 substitution in F170 (EU numbering) is the F170V or F170I substitution. In some embodiments, the HC CH1 substitution in S183 (EU numbering) is the S183L or S183I substitution. In some embodiments, the HC CH1 substitution in V185 (EU numbering) is the V185L substitution.In some embodiments, the HC CH1 substitution in C220 (EU numbering) is the C220S substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the E124C (lambda) substitution. In some embodiments, the LC CL1 substitution in position 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, the LC CL1 substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, HC CH1 includes or consists of substitutions for F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of substitutions for Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, HC CH1 includes the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC CL includes or consists of the substitutions E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, LC CL includes or consists of the substitutions E124C, L135F, and C214S (lambda) (EU numbering). In some embodiments, LC CL includes or consists of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering). In some embodiments, the antibody is human IgG. In some embodiments, the antibody is human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody.In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody.

[0072] In some embodiments, a method for producing an antibody or fragment thereof comprises providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), wherein HC1 CH1 contains or consists of substitutions at F126 and C220 (EU numbering), LC1 CL contains or consists of substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), HC2 CH1 contains or consists of substitutions at F170, S183, and V185 (EU numbering), and LC2 CL includes or consists of substitution L135 (EU numbering), HC1 CH1 and / or HC2 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V and V185A (EU numbering), LC1 CL and / or LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering). In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the HC1 CH1 substitution at F126 (EU numbering) is an F126C substitution, and the LC1 CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is an E124C (lambda) substitution. In some embodiments, the LC CL1 substitution at position 124 (EU numbering) is the Q124C (kappa) substitution. In some embodiments, the HC1 CH1 substitution at C220 (EU numbering) is the C220S substitution.In some embodiments, the LC1 CL substitution in C214 (EU numbering) is the C214S substitution. In some embodiments, the HC2 CH1 substitution in F170 (EU numbering) is the F170V or F170I substitution. In some embodiments, the HC2 CH1 substitution in S183 (EU numbering) is the S183L or S183I substitution. In some embodiments, the HC2 CH1 substitution in V185 (EU numbering) is the V185L substitution. In some embodiments, the LC2 CL substitution in L135 (EU numbering) is the L135F substitution. In some embodiments, HC1 CH1 includes or consists of F126C and C220S (EU numbering) substitutions, LC1 CL includes or consists of E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) substitutions (in some embodiments, LC CL includes or consists of E124C and C214S (lambda) substitutions, in some embodiments, LC CL includes or consists of Q124C and C214S (kappa) substitutions), HC2 CH1 includes or consists of F170I, S183L, V185L (EU numbering), and LC2 CL includes or consists of L135F (EU numbering) substitutions. In some embodiments, HC1 CH1 includes or consists of F126C and C220S (EU numbering) substitutions, LC1 CL includes or consists of E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) substitutions (in some embodiments, LC CL includes or consists of E124C and C214S (lambda) substitutions, in some embodiments, LC CL includes or consists of Q124C and C214S (kappa) substitutions), HC2 CH1 includes or consists of F170V, S183I, V185L (EU numbering), and LC2 CL includes or consists of L135F (EU numbering) substitutions. In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of SEQ ID NO: 9, and LC CL consists of substitutions in E18, L29, and C105 of SEQ ID NO: 1.In some embodiments, CH1 consists of substitutions in F9, F53, S66, V68, and C103 of sequence number 9, and LC CL consists of substitutions in Q17, L28, and C107 of sequence number 6. In some embodiments, the HC CH1 substitution in F9 is the F9C substitution. In some embodiments, the HC CH1 substitution in F53 is the F53V or F53I substitution. In some embodiments, the HC CH1 substitution in S66 is the S66L or S66I substitution. In some embodiments, the HC CH1 substitution in V68 is the V68L substitution. In some embodiments, the HC CH1 substitution in C103 is the C103S substitution. In some embodiments, the LC CL1 substitution in Q17 of sequence number 6 is the Q17C substitution. In some embodiments, the LC CL1 substitution in E18 of sequence number 1 is the E18C substitution. In some embodiments, the LC CL1 substitution in L28 of sequence number 6 is the L28F substitution. In some embodiments, the LC CL1 substitution at L29 in SEQ ID NO: 1 is the L29F substitution. In some embodiments, the LC CL1 substitution at C107 in SEQ ID NO: 6 is the C107S substitution. In some embodiments, the LC CL1 substitution at C105 in SEQ ID NO: 1 is the C105S substitution. In some embodiments, HC CH1 includes or consists of the substitutions F9C, F53V, S66I, V68L, and C103S, and LC CL includes the substitutions Q17C, L28F, and C107S. In some embodiments, HC CH1 contains or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL contains the E18C, L29F, and C105S substitutions in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and E18, L29, and C105 in SEQ ID NO: 1. In some embodiments, HC CH1 contains or consists of the F9C, F53V, S66I, V68L, and C103S substitutions, and LC CL contains the Q17C, L28F, and C107S substitutions in the positions corresponding to F9, F53, S66, V68, and C103 in SEQ ID NO: 9, and Q17, L28, and C107 in SEQ ID NO: 6. In some embodiments, the antibody is human IgG.In some embodiments, the antibody is a human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody.

[0073] In some embodiments, a method for producing an antibody or fragment thereof comprises providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), and the LC VL framework region (FR) and / or HC VH framework region (FR) comprising one or more substitutions. In some embodiments, the VL FR comprises substitutions of Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, the VH FR comprises substitutions of Q39 and / or G44 (EU numbering). In some embodiments, VH FR includes substitutions in Q39 and / or G44 (EU numbering), and VL FR includes substitutions in Q38 and / or G100 and / or G101 (EU numbering). In some embodiments, VL FR substitutions in Q38 include, or consist of, substitutions selected from the group consisting of Q38D and Q38K substitutions (EU numbering). In some embodiments, VL FR substitutions in G100 include, or consist of, substitutions selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering). In some embodiments, VL FR substitutions in G101 include, or consist of, substitutions selected from the group consisting of G101D and G101K substitutions (EU numbering). In some embodiments, VH FR substitutions in Q39 include, or consist of, substitutions selected from the group consisting of Q39D and Q39K substitutions (EU numbering). In some embodiments, the VH FR substitution in G44 includes or consists of substitutions selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering). In some embodiments, the VH FR substitution includes or consists of the Q39K substitution, and the VL FR substitution includes or consists of the Q38D substitution. In some embodiments, the VH FR substitution includes or consists of the Q39D substitution, and the VL FR substitution includes or consists of the Q38K substitution.In some embodiments, the substitution of VH FR includes or consists of G44K substitution, and the substitution of VL FR includes or consists of G100D substitution. In some embodiments, the substitution of VH FR includes or consists of G44D substitution, and the substitution of VL FR includes or consists of G100K substitution. In some embodiments, the substitution of VH FR includes or consists of G44C substitution, and the substitution of VL FR includes or consists of G100C substitution. In some embodiments, the substitution of VH FR includes or consists of G44K substitution, and the substitution of VL FR includes or consists of G101D substitution. In some embodiments, the substitution of VH FR includes or consists of G44D substitution, and the substitution of VL FR includes or consists of G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of the Q39K substitution, the substitution of LC1 VL FR includes or consists of the Q38D substitution, the substitution of HC2 VH FR includes or consists of the Q39D substitution, and the substitution of LC2 VL FR includes or consists of the Q38K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G100D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G100K substitution.In some embodiments, the substitution of HC1 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC2 VH FR includes or consists of G44C substitution, and the substitution of LC1 VL FR includes or consists of G100C substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of G44K substitution, the substitution of LC1 VL FR includes or consists of G101D substitution, the substitution of HC2 VH FR includes or consists of G44D substitution, and the substitution of LC2 VL FR includes or consists of G101K substitution. In some embodiments, the substitution of HC1 VH FR includes or consists of the G44D substitution, the substitution of LC1 VL FR includes or consists of the G101K substitution, the substitution of HC2 VH FR includes or consists of the G44K substitution, and the substitution of LC2 VL FR includes or consists of the G101D substitution.

[0074] In some embodiments, a method for producing an antibody or a fragment thereof comprises providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL), and the LC VL framework region (FR) and / or HC VH framework region (FR) comprising one or more substitutions. In some embodiments, HC1 or HC2 VH comprises or consists of any one of sequence numbers 53 to 64. In some embodiments, LC1 or LC2 VL comprises or consists of any one of sequence numbers 65 to 80. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 30, 43, 35, and 52, or SEQ ID NOs. 35, 52, 30, and 43, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 29, 44, 36, and 51, or SEQ ID NOs. 36, 51, 29, and 44, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequence of SEQ ID NOs. 28, 39, 33, and 48, or SEQ ID NOs. 33, 48, 28, and 39, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 27, 40, 34, and 47, or SEQ ID NOs. 34, 47, 27, and 40, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 26, 38, 31, and 45, or SEQ ID NOs. 31, 45, 26, and 38, respectively.In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 25, 37, 32, and 46, or SEQ ID NOs. 32, 46, 25, and 37, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 28, 41, 33, and 50, or SEQ ID NOs. 33, 50, 28, and 41, respectively. In some embodiments, each of the heavy and light chains of an HC1 / LC1 pair and each of the heavy and light chains of an HC2 / LC2 pair contains or consists of the sequences of SEQ ID NOs. 27, 42, 34, and 49, or SEQ ID NOs. 34, 49, 27, and 42, respectively. In some embodiments, HC1 includes or consists of sequence number 30, LC1 includes or consists of sequence number 43, HC2 includes or consists of sequence number 35, and LC2 includes or consists of sequence number 52. In some embodiments, HC1 includes or consists of sequence number 29, LC1 includes or consists of sequence number 44, HC2 includes or consists of sequence number 36, and LC2 includes or consists of sequence number 51. In some embodiments, HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 48. In some embodiments, HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 47. In some embodiments, HC1 includes or consists of sequence number 26, LC1 includes or consists of sequence number 38, HC2 includes or consists of sequence number 31, and LC2 includes or consists of sequence number 45. In some embodiments, HC1 includes or consists of sequence number 25, LC1 includes or consists of sequence number 37, HC2 includes or consists of sequence number 32, and LC2 includes or consists of sequence number 46.In some embodiments, HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 50. In some embodiments, HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 49. In some embodiments, HC1 includes or consists of sequence number 35, LC1 includes or consists of sequence number 52, HC2 includes or consists of sequence number 30, and LC2 includes or consists of sequence number 43. In some embodiments, HC1 includes or consists of sequence number 36, LC1 includes or consists of sequence number 51, HC2 includes or consists of sequence number 29, and LC2 includes or consists of sequence number 44. In some embodiments, HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 48, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 39. In some embodiments, HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 47, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 40. In some embodiments, HC1 includes or consists of sequence number 31, LC1 includes or consists of sequence number 45, HC2 includes or consists of sequence number 26, and LC2 includes or consists of sequence number 38. In some embodiments, HC1 includes or consists of sequence number 32, LC1 includes or consists of sequence number 46, HC2 includes or consists of sequence number 25, and LC2 includes or consists of sequence number 37. In some embodiments, HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 50, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 41.In some embodiments, HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 49, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 42.

[0075] In some embodiments, in addition to CL-CH1 pairing mutations F / JT7 or F / JT11, pairing mutations are introduced at the VL-CL interface, in addition to LC-HC pairing fidelity in the bispecific IgG1 molecule, optionally, if both Fabs have kappa LC as light chains. In some embodiments, residue pairs on the VL-VH interface are optionally mutated to Cys for disulfide bond formation or to residues of opposite charge for electrostatic interaction, as shown in Table 4. In some embodiments, the antibody or its binding fragment contains or consists of one or more of the substitution combinations shown in Table 4. For example, in some embodiments, the antibody or its binding fragment contains or consists of VH containing or consisting of Q39K, Q39D, G44K, G44D, or G44C substitutions. In some embodiments, the antibody or its binding fragment contains CH1 containing or consisting of F170V, S183I, or V185L substitutions. In some embodiments, the antibody or its binding fragment contains or comprises a VL comprising the Q38D, Q38K, G100D, G100K, or G100C substitution. In some embodiments, the antibody or its antigen-binding fragment contains or comprises a CL comprising the L135F substitution. In some embodiments, the antibody or its binding fragment contains or comprises a combination of substitutions listed for any one first Fab (cytokine C Fab) from V1, V2, V3, V4, V5, V6, V7, or V8 in Table 4. In some embodiments, the antibody or its binding fragment contains or comprises a combination of substitutions listed for any one second Fab (cytokine D Fab) from V1, V2, V3, V4, V5, V6, V7, or V8 in Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of a combination of substitutions listed for a first Fab and a second Fab from any one of V1, V2, V3, V4, V5, V6, V7, or V8 in Table 4.In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V2 of Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V3 of Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V4 of Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V5 of Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V6 of Table 4. In some embodiments, the antibody or its conjugated fragment contains or consists of the substitution combinations listed for the first Fab and / or second Fab in V7 of Table 4. In some embodiments, the antibody or its binding fragment includes or consists of a combination of substitutions listed for the first Fab and / or second Fab in V8 of Table 4. In some embodiments, the antibody or its antigen-binding fragment includes a first Fab containing VH with a Q39K substitution, wild-type CH1, a VL containing or consisting of a Q38D substitution, and wild-type CL, and / or a second Fab containing VH with a Q39D substitution, a CH1 containing or consisting of a Q38K substitution, a VL containing or consisting of a L135F substitution, and CL.In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab comprising VH containing a Q39D substitution, wild-type CH1, Q38K substitution, or a VL comprising the same, and wild-type CL, and / or a second Fab comprising VH containing a Q39K substitution, F170V, S183I, and / or V185L substitution, or a CH1 comprising the same, Q38D substitution, or a VL comprising the same, and a CL comprising the L135F substitution. In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab comprising VH containing a G44K substitution, wild-type CH1, G100D substitution, or a VL comprising the same, and wild-type CL, and / or a second Fab comprising VH containing a G44D substitution, F170V, S183I, and / or V185L substitution, or a CH1 comprising the same, G100K substitution, or a VL comprising the same, and a CL comprising the L135F substitution. In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab comprising VH with a G44D substitution, wild-type CH1, G100K substitution, or a VL comprising the same, and wild-type CL, and / or a second Fab comprising VH with a G44K substitution, F170V, S183I, and / or V185L substitution, or a CH1 comprising the same, G100D substitution, or a VL comprising the same, and L135F substitution, or a CL comprising the same. In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab comprising VH with a G44C substitution, wild-type CH1, G100C substitution, or a VL comprising the same, and wild-type CL, and / or a second Fab comprising wild-type VH, F170V, S183I, and / or V185L substitution, or a CH1 comprising the same, wild-type VL, and L135F substitution, or a CL comprising the same.In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab containing wild-type VH, wild-type CH1, wild-type VL, and wild-type CL, and / or a second Fab containing VH with a G44C substitution, F170V, S183I, and / or V185L substitution, or a CH1 consisting of the same, a G100C substitution, or a VL consisting of the same, and a CL consisting of the same. In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab containing VH with a G44K substitution, wild-type CH1, a G101D substitution, or a VL consisting of the same, and wild-type CL, and / or a second Fab containing VH with a G44D substitution, F170V, S183I, and / or V185L substitution, or a CH1 consisting of the same, a G101K substitution, or a VL consisting of the same, and a CL consisting of the same. In some embodiments, the antibody or its antigen-binding fragment comprises a first Fab comprising VH containing a G44D substitution, wild-type CH1, a VL containing or consisting of a G101K substitution, and wild-type CL, and / or a second Fab comprising VH containing a G44K substitution, a CH1 containing or consisting of a G101D substitution, or consisting of a G101D substitution, and a CL containing or consisting of a L135F substitution. In some embodiments, HC pairing in bispecific IgG is transmitted through a hole mutation (T366S / S368A / Y407V) in the HC of the first Fab (e.g., cytokine C) and a knob mutation (T366W) in the HC of the second Fab (e.g., cytokine D).

[0076] In some embodiments, the four strands (HC1, HC2, LC1, and LC2) are transfected into cells to be expressed in a ratio of 1:1:1:1. In some embodiments, the four strands (HC1, HC2, LC1, and LC2) are transfected into cells for expression in a ratio of 1:1:2:1. In some embodiments, the four strands (HC1, HC2, LC1, and LC2) are transfected into cells to be expressed in a ratio of 1:1:5:1. In some embodiments, the four strands (HC1, HC2, LC1, and LC2) are transfected into cells to be expressed in a ratio of 1:1:1.5:1.5. In some embodiments, four chains (HC1, HC2, LC1, and LC2) are transfected into cells so that the amounts of HC1, HC2, and LC2 are equal or nearly equal, and the amount of LC1 is expressed in a ratio of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 8, 9.5, 10 or more, or nearly that value, relative to HC1, HC2, and LC2. In some embodiments, the cells are Expi293 cells. In some embodiments, the IgG molecules expressed in the conditioned medium are captured on a protein A resin (e.g., MabSelect SuRe®). In some embodiments, protein A capture removes non-product-related impurities. In some embodiments, nonspecific binding is washed away with PBS (phosphate-buffered saline), and the expressed antibody is optionally eluted with 0.5% acetic acid, pH 3.5, and neutralized with 15% by volume of 1M Tris-HCl, pH 8.0 to a final pH of 7.4. In some embodiments, the antibody pool is further purified. In some embodiments, purification chromatography removes product-related impurities and residual impurities. In some embodiments, the antibody is purified, for example, by using mixed-mode chromatography. In some embodiments, a multimode cation exchanger (e.g., Capto MMC ImpRes®, CMM HyperCel, CIMmultus® PrimaS, CIMmultus® H-Bond) is used to purify the correctly paired antibody or its binding fragment (e.g., Fab).In some embodiments, the HC and LC of the bispecific antibody or its antigen-binding fragment are correctly paired in an amount (e.g., a percentage) that is either the value shown in Table 5, approximately that value (e.g., ±1, 5, or 10%), or at least that value. In some embodiments, the percentage of correctly assembled bispecific IgG1 is calculated by quantifying all present IgG1 species (excluding half-antibodies and homodimers of half-antibodies (see Table 5, e.g.)). For example, in some embodiments, the antibody or its antigen-binding fragment comprises cytokine C Fab containing Q39K, Q39D, G44C, or G44K substitutions or VH consisting thereof, wild-type CH1, Q38D, Q38K, G100C, or G101D substitutions or VL consisting thereof, and wild-type CL, and cytokine D Fab containing Q39D, Q39K, G44C, or G44D substitutions or VH consisting thereof, F170V, S183I, and / or V185L substitutions or CH1 consisting thereof, Q38K, Q38D, G100C, or G101K substitutions or VL consisting thereof, and L135F substitutions or CL consisting thereof, and correctly pairs with a probability of approximately 60-100%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V1, and the HC and LC of the bispecific antibody pair correctly in amounts of 55%, 60%, or 65%, approximately that%, or at least that%, optionally 55–65%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V2, and the HC and LC of the bispecific antibody pair correctly in amounts of 65%, 70%, or 75%, approximately that%, or at least that%, optionally 65–75%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V5, and the HC and LC of the bispecific antibody pair correctly in amounts of 90%, 95%, 99%, or 100%, approximately that%, or at least that%, optionally 90–100%.In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V6, and the HC and LC of the bispecific antibody correctly pair in amounts of 85%, 90%, or 95%, approximately that%, or at least that%, optionally 85–95%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V7, and the HC and LC of the bispecific antibody correctly pair in amounts of 45%, 50%, or 55%, approximately that%, or at least that%, optionally 45–55%. In some embodiments, purification, e.g., on a mixed-mode column (e.g., Capto MMC ImpRes®), removes mispaired contaminants and increases the amount (e.g., percent) of correctly paired bispecific antibody by 5%, 10%, 15%, 20%, 25%, or 30%, or at least that%, optionally 5–30%. In some embodiments, correct pairing is determined using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the antigen-binding fragment of the bispecific antibody correctly pairs in an amount (e.g., ±1, 5, or 10%) of the value shown in Table 6, or at least the value shown (e.g., as a percentage). For example, in some embodiments, the antibody or its antigen-binding fragment comprises cytokine C Fab containing Q39K, Q39D, G44C, or G44K substitutions or VH consisting thereof, wild-type CH1, Q38D, Q38K, G100C, or G101D substitutions or VL consisting thereof, and wild-type CL, and cytokine D Fab containing Q39D, Q39K, G44C, or G44D substitutions or VH consisting thereof, F170V, S183I, and / or V185L substitutions or CH1 consisting thereof, Q38K, Q38D, G100C, or G101K substitutions or VL consisting thereof, and L135F substitutions or CL consisting thereof, and correctly pairs with a probability of approximately 80-100%.In some embodiments, the bispecific antibody binding fragment includes the substitution combinations listed in Table 4 for V1, and the HC and LC of the bispecific antibody correctly pair in amounts of 80%, 85%, or 90%, approximately that%, or at least that%, optionally 80–90%. In some embodiments, the bispecific antibody or its binding fragment includes the substitution combinations listed in Table 4 for V2, and the HC and LC of the bispecific antibody correctly pair in amounts of 80%, 85%, or 90%, approximately that%, or at least that%, optionally 80–90%. In some embodiments, the bispecific antibody or its binding fragment includes the substitution combinations listed in Table 4 for V5, and the HC and LC of the bispecific antibody correctly pair in amounts of 90%, 95%, 99%, or 100%, approximately that%, or at least that%, optionally 90–100%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V6, and the HC and LC of the bispecific antibody correctly pair in amounts of 90%, 95%, 99%, or 100%, approximately that%, or at least that%, optionally 90–100%. In some embodiments, the bispecific antibody or its conjugated fragment includes the substitution combinations listed in Table 4 for V7, and the HC and LC of the bispecific antibody correctly pair in amounts of 80%, 85%, or 90%, approximately that%, or at least that%, optionally 80–90%. In some embodiments, purification, e.g., on a mixed-mode column (e.g., Capto MMC ImpRes®), removes mispaired contaminants and increases the amount (e.g., percent) of correctly paired bispecific antibody by 5%, 10%, 15%, 20%, 25%, or 30%, or at least that%, optionally 5–30%. In some embodiments, the correct pairing is determined using liquid chromatography-mass spectrometry (LC-MS).

[0077] In some embodiments, the antibody heavy chain or its antigen-binding fragment pairs correctly with probabilities as shown in Table 7. For example, in some embodiments, an antibody or its antigen-binding fragment containing a first Fab and / or a second Fab pairs correctly with approximately 60–100% probability when transfected with DNA encoding the light chains of the first Fab and the second Fab in a ratio of 1:1, 2:1, or 5:1. In some embodiments, the first HC and the second HC are present in equal amounts, and the HC1:HC2:LC1:LC2 ratios are 1:1:1.5:1.5, 1:1:2:1, and 1:1:2.5:0.5. In some embodiments, the correct pairing of the first Fab (HC1 / LC1) can be increased by increasing the ratio of the first Fab (LC1) light chain to the second Fab (LC2) light chain from 1:1 to 2:1 or 5:1. In some embodiments, increasing the ratio of the first Fab(LC1) light chain to the second Fab(LC2) light chain from 1:1 to 2:1 or 5:1 removes up to approximately 80-100% of HC1 / LC2 mispairings. In some embodiments, increasing the LC1:LC2 ratio results in an increase in mispairings (HC2 / LC1). In some embodiments, purification, e.g., purification using a mixed-mode column (e.g., Capto MMC ImpRes®), removes up to approximately 80-100% of HC2 / LC1 mispairing contaminants.

[0078] In some embodiments, multiple antibodies containing two heavy / light chain pairs are produced, and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibodies containing two heavy / light chain pairs produced contain HC1 / LC1 and HC2 / LC2, or antibodies containing HC1 / LC1 and HC2 / LC2 pairs are produced in a percentage within the range defined by any two of the aforementioned values. For example, in some embodiments, about 50-100, 50-95, 50-90, 50-80, 50-75, 50-70, 50-60, 50-55, 55-100, 55-95, 55-90, 55-80, 55-75, 55-70, 55-60, 60-100, 60-95, 60-90, 60-80, 60-75, 60-70, 70-100, 70-95, 70-90, 70-80, 75-100, 75-95, 75-90, 75-80, 80-100, 80-95, 80-90, 90-100, 90-95, or 95-100% of the produced antibodies consist of HC1 / LC1 pairs and HC2 / LC2 pairs.

[0079] In some embodiments, 0% or less of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% of the antibody containing two heavy / light chain pairs produced includes either HC1 / LC2 or HC2 / LC1, or includes a percentage of HC1 / LC2 pairs or HC2 / LC1 pairs within the range defined by any two of the aforementioned values. For example, in some embodiments, the composition contains about 0.1-50, 0.1-40, 0.1-20, 0.1-25, 0.1-20, 0.1-10, 0.1-5, 0.1-1, 1-50, 1-40, 1-30, 1-25, 1-20, 1-20, 1-5, 5-50, 5-40, 5-30, 5-25, 5-10, 10-50, 10-40, 10-25, 25-50, 25-40, 25-30, 30-50, 30-40, or 40-50% of HC1 / LC2 pairs or HC2 / LC1 pairs. In some embodiments, the percentage of antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs refers to the percentage of HC1 / LC1 pairs and HC2 / LC2 pairs before any enrichment of the antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs. In some embodiments, the percentage (%) of antibody containing HC1 / LC2 pairs and HC2 / LC1 pairs refers to the percentage of HC1 / LC2 pairs and HC2 / LC1 pairs before any enrichment of the antibody containing HC1 / LC1 pairs and HC2 / LC2 pairs.

[0080] In some embodiments, providing includes providing one or more polynucleotides encoding HC1, HC2, LC1, and LC2. In some embodiments, one or more polynucleotides encode IgG Fab or an antibody. In some embodiments, the polynucleotide encodes human IgG. In some embodiments, the polynucleotide encodes human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the polynucleotide encodes a chimeric antibody. In some embodiments, the polynucleotide encodes a humanized antibody. In some embodiments, the polynucleotide encodes a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody.

[0081] In some embodiments, providing includes providing an expression vector or construct encoding HC1, HC2, LC1, and LC2. In some embodiments, the expression vector or construct encodes HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:2:1, or 1:1:5:1. In some embodiments, the expression vector is a plasmid or viral vector. In some embodiments, the expression vector or construct encodes IgG Fab or an antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a human antibody or a fragment thereof. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG. In some embodiments, the expression vector or construct expresses a polynucleotide encoding human IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a humanized antibody. In some embodiments, the expression vector or construct expresses a polynucleotide encoding a chimeric or humanized IgG1, IgG2, IgG3, or IgG4 antibody.

[0082] In some embodiments, providing includes providing cells expressing HC1, HC2, LC1, and LC2. In some embodiments, the cells express IgG Fab or antibodies. In some embodiments, the cells are mammalian cells or bacterial cells. In some embodiments, the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells. In some embodiments, the cells include one or more polynucleotides encoding a human antibody or a fragment thereof, an expression vector, or a construct. In some embodiments, the fragment includes an antigen-binding fragment. In some embodiments, the cells include one or more polynucleotides encoding human IgG, an expression vector, or a construct. In some embodiments, the cells include one or more polynucleotides encoding human IgG1, IgG2, IgG3, or IgG4 antibodies, an expression vector, or a construct. In some embodiments, the cells include one or more polynucleotides encoding a chimeric antibody, an expression vector, or a construct. In some embodiments, the cells include one or more polynucleotides encoding a humanized antibody, an expression vector, or a construct.

[0083] In some embodiments, the method further comprises extraction and / or purification of an antibody or its fragment. In some embodiments, the antibody or its fragment is extracted and / or purified from cell supernatant or cell lysate. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a human antibody or its fragment. In some embodiments, the fragment comprises an antigen-binding fragment. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding human IgG1, IgG2, IgG3, or IgG4 antibodies. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a chimeric antibody. In some embodiments, the cell comprises one or more polynucleotides, expression vectors, or constructs encoding a polynucleotide encoding a humanized antibody. In some embodiments, one or more polynucleotides encode LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately in a ratio, optionally 1:5, or approximately 1:5. In some embodiments, one or more polynucleotides encode HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, optionally 1:1, 1.5:1, or 2:1, or approximately in a ratio. In some embodiments, one or more polynucleotides encode HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately in ratio. In some embodiments, one or more polynucleotides encode HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately in ratio. In some embodiments, one or more polynucleotides encode IgG Fab or an antibody.In some embodiments, one or more expression vectors or constructs encode HC1, HC2, LC1, and LC2. In some embodiments, cells express HC1, HC2, LC1, and LC2, and optionally, one or more polynucleotides and / or vectors encode HC1, HC2, LC1, and LC2. In some embodiments, cells express LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately, and optionally, the ratio is 1:5 or approximately 1:5. In some embodiments, cells express HC1 and HC2 in ratios of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or optionally in ratios of 1:1, 1.5:1, or 2:1, or approximately in ratio. In some embodiments, cells express HC1 and LC2 in ratios of 1:0.5 to 1:3, or optionally in ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately in ratio. In some embodiments, cells express HC1, HC2, LC1, and LC2 in ratios of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately in ratio. In some embodiments, the cells express IgG Fab or an antibody. In some embodiments, the cells are mammalian cells or bacterial cells. In some embodiments, the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells. In some embodiments, the method includes extraction and / or purification of the expressed antibody or its fragments. In some embodiments, the expressed antibody or its fragments are extracted and / or purified from the cell supernatant or cell lysate. [Examples]

[0084] (Example 1) The Fab structure 7REW from the PDB was visualized using the open-source software Pymol. The interaction interface between CL and CH1 in 7REW was identified, and the residues in the CL and CH1 domains that contribute most to Fab domain packing stability were identified as L135(CL), F170(CH1), S183(CH1), and V185(CH1) based on EU numbering (L135 in LC and F176, S189, and V191 in HC based on AA sequence number numbering in 7REW).

[0085] The design criteria for specific LC-HC pairings were as follows: 1) Mutant LC and LC should be packed similarly to wild-type LC and HC interactions in the CL and CH1 domains. 2) Mutant HC-wt LC interactions and mutant LC-wt HC interactions should not be supported (side-chain steric collision). The mutant sets were assembled based on EU numbering with L135(CL) and F170, S183, V185(CH1) (based on AA sequence number numbering in 7REW, the numbering is L135 for LC and F176, S189, and V191 for HC). The final mutant sets that met the design criteria, according to AA sequence number numbering in 7REW, are summarized in Table 1.

[0086] [Table 1]

[0087] These mutations were introduced into the CL and CH1 domains of the antibody whose structure is represented by PDB 7REW.

[0088] Figure 4 is an exemplary representation of an embodiment of pairing analysis of a WT heavy chain and WT light chain in the presence of a mutant light chain containing L135F.

[0089] Expression plasmids encoding LCs and HCs of the 7REW antibody were co-expressed in HEK293 cells via transient transfection in the presence of mutant LCs including E124C, L135F, and C214S. Packing energy calculations in Table 1 suggested that mutant LCs including L135F did not pair with wt HCs, resulting in inaccurate wild-type LC and HC pairing. Intact, correctly paired LC and HC assemblies were analyzed using SDS-PAGE under non-reducing conditions or using reversed-phase HPLC on a biphenyl column with water / acetonitrile (0.1% TFA) as the mobile phase. Inaccurately paired HCs and mutant LCs (L135F, C214S) appeared as one or more LCs lacking IgG under SDS-PAGE or RPHPLC, but these analyses were performed under denaturing conditions that allowed for the separation of subunits when they were not covalently bonded.

[0090] Figure 5 is an exemplary representation of an embodiment of pairing analysis of mutant heavy chains paired with mutant WT light chains in the presence of WT light chains.

[0091] In addition to expression plasmid pairs encoding LCs and HCs with the mutation sets listed in Table 1, further LC mutations of E124C and C214S (lambda), HC mutations of F126C and C220S, and WT LCs were co-expressed in HEK293 cells via transient transfection. The E124C and C214S LC mutations and the F126C and C220S HC mutations provided readout of mutant LC / HC pairings via disulfide crosslinking between E124C in LC and F126C in HC. Mispaired wt LCs and mutant HCs lacked disulfide bond linkage due to the C2220S mutation in mutant HC. Correct assembly of LCs and HCs was analyzed using SDS-PAGE under non-reducing conditions or using reversed-phase HPLC on a biphenyl column with water / acetonitrile (0.1% TFA) as the mobile phase. Mutant HCs that mismatched with wt LCs appeared as one or more IgG-deficient LCs under SDS-PAGE or RP-HPLC, but these analyses were performed under denaturing conditions that allowed for the separation of subunits when they were not covalently bonded.

[0092] Figure 6A is a table showing the identity of the sample embodiments in each lane of the SDS-page gel in Figure 6B.

[0093] Figure 6B shows SDS-PAGE gels of different HC / LC pairing embodiments under non-reducing conditions. Mutation sets that enhance LC / HC pairing specificity enhance the percentage of correctly assembled intact antibodies (upper bands of the two LCs and two HCs) in the presence of similar amounts of competing, unchanged wt LC-HC-JT7 or JT11 paired with LC-F, and achieve equivalent correct assembly of wt IgG in the presence of competing wt LCs.

[0094] Figure 7A is a table showing the identity of the sample embodiments in each lane of the SDS-page gel in Figure 7B.

[0095] Figure 7B shows SDS-page gels representing different embodiments of HC / LC pairing under reducing conditions.

[0096] In addition to the expression plasmid pairs encoding LC and HC with the mutation sets listed in Table 1, further LC mutations E124C and C2124S, HC mutations F126C and C220S, and wt LC were co-expressed in 293 cells via transient transfection as shown in Figure 5. IgG molecules during acclimatization were analyzed for titer and LC-HC pairing using protein A purification analysis.

[0097] During transient expression of IgG molecules, wt LC and mutant LC were co-transfected in equal amounts to provide readout of mutant LC / HC pairing specificity. When mutant LC paired with mutant HC, the IgG molecule assembled correctly and moved on SDS-PAGE as an intact IgG band under non-reducing conditions. Mispaired wt LC and mutant HC did not form a disulfide bond, resulting in the detection of an IgG band lacking LC under non-reducing conditions (a lower band). The mutant sets listed in Table 1 are represented in lanes 1-18. Through this analysis, two mutant sets (F / JT7 and F / JT11) exhibiting the desired pairing specificity were identified.

[0098] Figure 8 shows a series of chromatograms of embodiments of RP-HPLC quantification of correct LC-HC pairings in positive controls and selected mutant sets.

[0099] As shown in Figure 8, proper LC-HC pairing in F / JT7 and F / JT11 mutants occurs at least as frequently as in wt IgG in the presence of interfering wt LC.

[0100] Table 2 shows the appropriate quantification of LC-HC pairings.

[0101] [Table 2]

[0102] As is clear from Table 2, proper LC-HC pairing occurs in F / JT7 and F / JT11 mutants at least as frequently as in wt IgG.

[0103] Figure 9A is an illustrative representation of an embodiment of the correct pairing of the heavy and light chains of the JT7 / F and JT11 / F dimerized pairs in a bispecific antibody.

[0104] Figure 9B is a chromatogram of an embodiment of purification and correct assembly analysis by RP-HPLC for purified bispecific antibodies using an LC / HC pairing strategy for selective disulfide (R2), JT7 / F, and JT11 / 11 dimerized pairs.

[0105] Figure 9C shows an SDS-page gel of an embodiment of a purified bispecific antibody based on the JT7 / F and JT11 / F dimerization mutation set under reducing and non-reducing conditions.

[0106] A plasmid encoding bispecific MAB-R2:Fab1 from LC1-wt kappa LC, HC1-wt HC with a whole mutation (T366S, L368A, Y407V, EU numbered), Fab2 from LC2-lambda LC with E124C, C214S (EU numbered), and HC2-(F126C, C220S) with a knob mutation (S366W, EU numbered) was transiently co-transfected into HEK293 cells. The individual subunits were assembled in HEK293 cells as bispecific MAB--R2 bispecific antibodies and associated misassembled impurities. The bispecific MAB--R2 was used as a reference benchmark bispecific antibody in accordance with U.S. Patent No. 10,344,099(B2), in which LC-HC specific pairing is transmitted by a selective disulfide bond between the CL domain of the LC and the CH1 domain of the HC (E124C in the LC linked to F126C).

[0107] A plasmid encoding a bispecific MAB--JT7:LC1-wt kappa LC-derived Fab1, HC1-wt HC with a whole mutation (T366S, L368A, Y407V, EU numbered), LC2-lambda LC-derived Fab2 with L135F (EU numbered), and HC2- (JT7 mutation set) with a knob mutation (S366W, EU numbered) was transiently co-transfected into HEK293 cells. The individual subunits were assembled in HEK293 cells as bispecific MAB--JT7 bispecific antibodies and associated misassembled impurities.

[0108] A plasmid encoding the bispecific MAB--JT11: Fab1 from LC1-wt kappa LC, HC1-wt HC with a whole mutation (T366S, L368A, Y407V, EU numbered), Fab2 from LC2-lambda LC with L135F (EU numbered), and HC2- (JT11 mutation set) with a knob mutation (S366W, EU numbered) was transiently co-transfected into HEK293 cells. The individual subunits were assembled in HEK293 cells as the bispecific MAB--JT7 bispecific antibody and associated misassembled impurities.

[0109] Bispecific MAB-R2, bispecific MAB-JT7, and bispecific MAB-JT11 were captured from conditioned medium of 293 transfected cells using a Mabselect PrismA (Protein A column). Impurities from the transfection host were removed by washing with phosphate-buffered saline. Binding IgG was recovered by acidic elution using 87 mM acetate, pH 3.5. The antibody-containing fractions were pooled and their pH was adjusted to pH 5.0. These pooled fractions are called the ProA pools. The ProA pools of bispecific MAB-R2, bispecific MAB-JT7, and bispecific MAB-JT11 were further purified using a Capto-MMC ImpRes (Cytiva) multimode chromatography column with pH gradient elution from pH 5.0 to pH 8.9. The Capto-MMC fractions were analyzed by SDS-PAGE, and combined with the correctly assembled bispecific antibody fractions, biospecific activity and molecular assembly were analyzed.

[0110] Figure 10A is a graph illustrating the neutralizing activity of the bispecific molecule Fab Arm 1 in HEK-293 reporter cells.

[0111] Human cytokine A blockade assay using a bispecific antibody in HEK-293 reporter cells. Binding of cytokine A to its receptor complex activates downstream signaling pathways, resulting in cytokine A-inducible expression of luciferase in HEK-293 reporter cells. To analyze cytokine neutralization activity, reporter HEK-293 cells were co-incubated with 10 ng / mL of human cytokine A and a bispecific antibody at 37°C for 5 hours. Bioluminescence signals were measured after 5 hours of co-incubation. Data are presented using the percentage of cytokine-luciferase inhibition.

[0112] Figure 10B is a graph illustrating the neutralizing activity of bispecific molecules in Fab Arm 2 of a cytokine B blockade assay using a bispecific antibody in HEK-293 reporter cells.

[0113] Human cytokine B blockade assay using a bispecific antibody in HEK-293 reporter cells. Binding of cytokine B to its receptor complex activates downstream signaling pathways, resulting in alkaline phosphatase expression. Here, the 293 cell line, containing the cytokine B receptor complex on its cell surface, was stably transfected with secreted embryonic alkaline phosphatase (SEAP) under the control of the cytokine B signaling pathway. The cytokine B signaling pathway was monitored using the HEK-293 reporter cell line by measuring SEAP activity in conditioned medium. In the cytokine B neutralization assay, HEK-293 reporter cells were co-incubated with 10 ng / mL of human cytokine B and a bispecific antibody at 37°C for 5 hours. After 5 hours of co-incubation, bioluminescence signals were measured. Data are presented using the percentage of SEAP activity inhibition.

[0114] Figure 11 shows that the combination of mutation sets JT11 / F or JT7 / F with selective disulfide R2 is beneficial for the molecular assembly of bispecific antibodies and their expression yields.

[0115] Figure 11A shows SDS-PAGE analysis under reducing and non-reducing conditions for different conditioned medium embodiments of HEK-293 cells transfected with different plasmids encoding bispecific antibodies HC and LC.

[0116] Figure 11B is a table showing the quantification of the expression and capture yield of bispecific antibodies in different embodiments.

[0117] Figure 11C is a set of chromatograms representing embodiments of RP-HPLC quantification of correct LC-HC pairings of plasmid-produced JT7 and JT11 dimerization pairs.

[0118] Bispecific MAB--R2: A reference benchmark bispecific antibody based on the technology from U.S. Patent No. 10,344,099(B2), encoding plasmids containing Fab1 from LC1-wt kappa LC, HC1-wt HC with whole mutations (T366S, L368A, Y407V, EU numbered), Fab2 from LC2-lambda LC with E124C, C214S (EU numbered), and HC2-(F126C, C220S) with a knob mutation (S366W, EU numbered).

[0119] Bispecific MAB--R2-JT7: A plasmid encoding Fab1 from LC1-wt kappa LC, HC1-wt HC with hole mutations (T366S, L368A, Y407V, EU numbered), Fab2 from LC2-lambda LC with E124C and L135F, C214S (EU numbered), and HC2-(F126C, F170I, S183L, V185L, C220S) with a knob mutation (S366W, EU numbered).

[0120] Bi-specific MAB--R2-JT11: A plasmid encoding Fab1 from LC1-wt kappa LC, HC1-wt HC with whole mutations (T366S, L368A, Y407V, EU numbered), Fab2 from LC2-lambda LC with E124C and L135F, C214S (EU numbered), and HC2-(F126C, F170V, S183I, V185L, C220S) with a knob mutation (S366W, EU numbered).

[0121] The bispecific MAB--R2, bispecific MAB--R2-JT7, and bispecific MAB--R2-JT11 were transfected into 293 cells. These bispecific antibodies in conditioned medium from the transfected 293 cells were captured by Mabselect PrismA (Protein A column). The ProA pool was analyzed for correct bispecificity assembly by SDS-PAGE and RP-HPLC. The percentage of correctly assembled bsMab was estimated from intact bsMab peaks on RP-HPLC (Figure 11C).

[0122] Mutation sets F / JT7 (L135 in CL, and F170I, S183L, and V185L in CH1, EU numbered) and F / JT11 (L135 in CL, and F170V, S183I, and V185L, EU numbered) (Figure 6 as an example), which enhance LC / HC pairing specificity in antibodies containing lambda LC, were introduced into Fab structures (Fab in PDB 1A4J) containing kappa LC as light chains. The corresponding mutation sets in kappa Fab (PDB 1A4J) compared to wild-type Fab are summarized in Table 3.

[0123] [Table 3]

[0124] Figure 17A is a bar graph showing the quantification of bispecific antibody expression in CHO cells.

[0125] Figure 17B is a bar graph showing the quantification of the ProA pool analyzed by capillary electrophoresis of sodium dodecyl sulfate (CE-SDS).

[0126] Figure 17C is a bar graph illustrating an embodiment of quantification of mismatch species by mass spectrometry.

[0127] CHO cells transfected with DNA encoding 2HC and 2LC, bispecific antibodies against cytokine A and cytokine B, were plated in MSX selection medium as 20,000 cell / well minipools in 96-well plates. Minipools stably expressing the bispecific molecules could be harvested two weeks after MSX selection, and the titers of the bispecific antibodies were screened. The top eight minipools were subjected to a 14-day cell culture production assay. As shown in Figure 17A, the titers of the top eight minipools ranged from 2.98 to 4.9 g / L.

[0128] The conditioned media from the top eight bispecific CHO minipools of antibodies against cytokines A and B were captured on protein A resin-MabSelect SuRe. Nonspecific binding was washed away with PBS (phosphate-buffered saline) solution. The expressed antibodies were eluted with 0.5% acetic acid, pH 3.5, and neutralized with 15% by volume of 1M Tris-HCl, pH 8.0, to a final pH of 7.4. The neutralized protein A eluate was called the ProA pool.

[0129] The ProA pools from the top eight minipools were analyzed by capillary electrophoresis using sodium dodecyl sulfate (CE-SDS). The main peak indicated that expressed antibodies were recovered as intact antibody species. The semi-antibodies were those correctly paired against cytokine B (Fab against cytokine B on Fc without correctly formed hinged disulfide).

[0130] The ProA pool was digested using FabALACTICA (Genovis) to separate Fab from Fc fragments. Fab was analyzed by LC-MS to correct for pairing percentages (Figure 17C). Mismatched species were calculated as a percentage of (X+Y) / total Fab. X = HC_cytokine A paired with LC_cytokine B. Y = HC_cytokine B paired with LC_cytokine A. Total Fab = correct Fab_cytokine_A + correct Fab_cytokine_B + X + Y.

[0131] (Example 2) To ensure LC-HC pairing fidelity in the bispecific IgG1 molecule, particularly when both Fabs have kappa LC as light chains, pairing mutations were introduced at the VL-CL interface in addition to the CL-CH1 pairing mutations F / JT7 or F / JT11. Residue pairs on the VL-VH interface were mutated to Cys for disulfide bond formation or to residues with opposite charges for electrostatic interaction (Table 4).

[0132] [Table 4]

[0133] HC pairing in bispecific IgG transmitted a knob mutation in the HC of cytokine D (T366W) and a whole mutation in the HC of cytokine C (T366S / S368A / Y407V).

[0134] To screen for mutations that improve pairing specificity, HC and k-LC variants of cytokines C and D (Table 5) were co-transfected into Expi293 cells with a four-chain plasmid ratio of 1:1:1.5:1.5 (HC1:HC2:LC1:LC2). IgG molecules expressed in conditioned medium were captured on protein A resin-MabSelect SuRe. After washing away nonspecific binding with PBS (phosphate-buffered saline), the expressed antibodies were eluted with 0.5% acetic acid, pH 3.5, and neutralized with 15% by volume of 1M Tris-HCl, pH 8.0, to a final pH of 7.4. Purified IgG molecules from the conditioned medium were then analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0135] The pool was further purified using a mixed-mode column, Capto MMC ImpRes. The fractions were pooled separately as bispecific antibody and mispair pools and analyzed by LC-MS.

[0136] Correctly bispecific antibodies and misassembled IgG from the ProA pool and MMC pool were first treated with PNGase to generate deglycosylated, intact proteins. The deglycosylated, intact molecules were analyzed by LC-MS to identify mispairing impurities. The percentage of correctly assembled BsIgG1 was calculated by quantifying all present IgG1 species (excluding half-antibodies and homodimers of half-antibodies (Table 5)). IgG molecules with LCs that pair to both HCs were labeled as common cytokine D LC or common cytokine C LC mispairing impurities. The ProA and MMC pools of IgG molecules were also treated with FabALACTICA (Genovis) to release Fab fragments from the IgG molecules. The percentage of mispairing in each Fab-containing arm was also determined by LC-MS (Table 6). Tables 5 and 6 both show that the second purification step, the Capto MMC ImpRes column, did not successfully remove mispairing contaminants when the LC of cytokine D paired with the HC of both cytokine D and cytokine C.

[0137] [Table 5]

[0138] [Table 6]

[0139] To demonstrate the robustness of pairing mutations in the assembly and production processes of bispecific IgG, HEK293 cells were transfected with different amounts of cytokine C (LC-C) and cytokine D (LC-D) for the V2 construct at 1:1, 2:1, and 5:1 ratios, respectively. Cytokine C (HC-C) and cytokine D (HC-D) were present in equal amounts, with ratios of HC-C, HC-D, LC-C, and LC-D being 1:1:1.5:1.5, 1:1:2:1, and 1:1:2.5:0.5, respectively. IgG in conditioned medium was first captured on protein A resin MabSelect SuRe, eluted, and then purified in a second step on a Capto MMC ImpRes column. The ProA or MMC-purified protein pool was treated with FabALACTICA (Genovis) to release Fab fragments, which were analyzed by LC-MS to determine the percentage of HC / LC mispairings (Table 7).

[0140] [Table 7]

[0141] Figure 18A is a chromatogram showing an embodiment of the quantification of Fab fragment pairings analyzed by LC-MS.

[0142] Figure 18B is a chromatogram showing an example of quantification of purified Fab fragment pairings, analyzed by LC-MS.

[0143] The results indicate that increased LC-C expression reduced common LC-D mispairing and increased common LC-C mispairing. Purification in the second step, which removed LC-C mispairing contaminants, increased the percentage of correctly assembled BsIgG in the final purified product.

Claims

1. An antigen-binding fragment (Fab), The human IgG heavy chain (HC) variable domain (VH), the human IgG heavy chain (HC) constant domain (CH1), the human IgG light chain (LC) variable domain (VL), and the human IgG light chain (LC) constant domain (CL) are included, wherein the HC CH1 includes substitutions in F170, S183, and V185 (EU numbering), the LC CL includes substitution in L135 (EU numbering), and the HC1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). The CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (European numbering).

2. The Fab according to claim 1, wherein CH1 consists of substitutions in F170, S183, and V185 (EU numbering), and CL consists of substitutions in L135 (EU numbering).

3. The Fab according to any one of claims 1 to 2, wherein the HC CH1 substitution in F170 (EU numbering) is F170I or F170V substitution.

4. The Fab according to any one of claims 1 to 3, wherein the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution.

5. The Fab according to any one of claims 1 to 4, wherein the HC CH1 substitution in V185 (EU numbering) is a V185L substitution.

6. The Fab according to any one of claims 1 to 5, wherein the LC CL1 substitution in L135 (EU numbering) is an L135F substitution.

7. The Fab according to claim 1 or 2, wherein the HC CH1 includes or consists of the F170V, S183I, and V185L (EU numbering) substitutions, and the LC CL includes or consists of the L135F (EU numbering) substitution.

8. The Fab according to claim 1 or 2, wherein the HC CH1 includes or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and the LC CL includes or consists of the L135F (EU numbering) substitution.

9. An antigen-binding fragment (Fab), The human IgG heavy chain (HC) variable domain (VH), the human IgG heavy chain (HC) constant domain (CH1), the human IgG light chain (LC) variable domain (VL), and the human IgG light chain (LC) constant domain (CL) are included, wherein the HC CH1 includes substitutions of F126, F170, S183, V185, and C220 (EU numbering), and the LC CL includes substitutions of E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and optionally, the LC CL includes substitutions of E124, L135, and C214 (lambda), or optionally, the LC CL includes substitutions for Q124, L135, and C214 (kappa), while HC1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). The CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

10. The Fab according to claim 9, wherein CH1 consists of substitutions in F126, F170, S183, V185, and C220 (EU numbering), and LC CL includes substitutions in E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), optionally, LC CL includes substitutions in E124, L135, and C214 (lambda), or optionally, LC CL includes substitutions in Q124, L135, and C214 (kappa).

11. The Fab according to claim 9 or 10, wherein the HC CH1 substitution in F126 (EU numbering) is an F126C substitution.

12. The Fab according to any one of claims 9 to 11, wherein the HC CH1 substitution in F170 (EU numbering) is F170V or F170I substitution.

13. The Fab according to any one of claims 9 to 12, wherein the HC CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.

14. The Fab according to any one of claims 9 to 13, wherein the HC CH1 substitution in V185 (EU numbering) is a V185L substitution.

15. The Fab according to any one of claims 9 to 14, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution.

16. The Fab according to any one of claims 9 to 15, wherein the LC CL substitution in E124 (lambda) or Q124 (kappa) (EU numbering) is E124C or Q124C substitution, and optionally, the LC CL substitution in E124 (lambda) is E124C substitution, or optionally, the LC CL substitution in Q124 (kappa) is Q124C substitution.

17. The Fab according to any one of claims 9 to 16, wherein the LC CL substitution in L135 (EU numbering) is an L135F substitution.

18. The Fab according to any one of claims 9 to 17, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution.

19. The Fab according to claim 9, wherein the HC CH1 includes or comprises the substitutions F126C, F170V, S183I, V185L, and C220S (EU numbering), and the LC CL includes the substitutions E124C, L135F, and C214S (lambda) or Q124C, L135F, and C214S (kappa) (EU numbering), and optionally, the LC CL includes the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally, the LC CL includes the substitutions Q124C, L135F, and C214S (kappa) (EU numbering).

20. The Fab according to claim 9, wherein the HC CH1 includes or comprises the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and the LC CL includes the substitutions E124C, L135F, and C214S (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and optionally, the LC CL includes the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally, the LC CL includes the substitutions Q124, L135, and C214 (kappa) (EU numbering).

21. An antigen-binding fragment (Fab), The human IgG heavy chain (HC) variable domain (VH), the human IgG heavy chain (HC) constant domain (CH1), the human IgG light chain (LC) variable domain (VL), and the human IgG light chain (LC) constant domain (CL) are included, wherein the HC CH1 includes substitutions of F126 and C220 (EU numbering), and the LC CL includes substitutions of E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering), optionally, the LC CL includes substitutions of E124 and C214 (lambda) (EU numbering), or optionally, the LC CL includes substitutions of Q124 and C214 (kappa) (EU numbering). The aforementioned HC1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170I, F170M, F170Y, F170S, F170A, F170V, S181I, S181T, S181M, S183A, S183L, S183I, S183V, V185L and V185A (EU numbering), The CL is an antigen-binding fragment (Fab) that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (European numbering).

22. The Fab according to claim 21, wherein the HC CH1 substitution in F126 (EU numbering) is an F126C substitution, the LC CL substitution in position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC CL substitution in position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC CL substitution in position 124 (EU numbering) is a Q124C (kappa) substitution.

23. The Fab according to any one of claims 21 to 22, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution.

24. The Fab according to any one of claims 21 to 23, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution.

25. The Fab according to any one of claims 1 to 24, wherein the HC CH1 includes or consists of sequence number 15 or 19, the LC CL includes or consists of sequence number 2 or 7, or the HC CH1 includes or consists of sequence number 23 or 24, and the LC CL includes or consists of sequence number 5 or 8.

26. An antibody comprising the Fab described in any one of claims 1 to 25.

27. The antibody according to claim 26, further comprising a second Fab, wherein one of the Fabs, but not both, comprises HC CH1 with substitutions at F126 and C220 (EU numbering), and LC CL with substitutions at E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering), optionally, LC CL comprising substitutions at E124 and C214 (lambda) (EU numbering), or optionally, LC CL comprising substitutions at Q124 and C214 (kappa) (EU numbering).

28. The antibody according to claim 27, wherein the HC CH1 substitution at F126 (EU numbering) is an F126C substitution, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC CL substitution at position 124 (EU numbering) is a Q124C (kappa) substitution.

29. The antibody according to any one of claims 27 to 28, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution.

30. The antibody according to any one of claims 27 to 29, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution.

31. An antibody comprising one or more IgG Fabs having wild-type CH and wild-type CL, and one or more Fabs according to any one of claims 1 to 30.

32. The antibody comprises the Fab described in any one of claims 1 to 8 and the Fab described in any one of claims 21 to 24, or the Fab described in any one of claims 9 to 20, as well as a Fab that does not contain HC CH1 with substitutions at F126 and C220 (EU numbering), and a Fab that does not contain LC CL with substitutions at position 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), optionally wherein LC CL includes substitutions at position E124 and C214 (EU numbering) for lambda, or optionally wherein LC CL includes substitutions at position Q124 and C214 (EU numbering) for kappa, as described in any one of claims 27 to 31.

33. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), Each HC1 and HC2 comprises the heavy chain variable domain (VH) and the first heavy chain constant domain (CH1) of human IgG, and each LC1 and LC2 comprises the light chain variable domain (VL) and the light chain constant domain (CL) of human IgG. The HC1 CH1 includes substitutions for F170, S183, and V185 (EU numbering), the LC1 CL includes substitutions for L135 (EU numbering), and the HC1 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering). The LC1 CL is an antibody or its fragment that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

34. The antibody or fragment thereof according to claim 33, wherein the HC1 CH1 consists of substitutions in F170, S183, and V185 (EU numbering), and the LC1 CL consists of substitutions in L135 (EU numbering).

35. The antibody or fragment according to claim 33, wherein the HC2 CH1 includes or consists of SEQ ID NO: 9, and the LC2 CL includes or consists of SEQ ID NO: 1 or 6.

36. The antibody or fragment thereof according to claim 33, wherein the HC1 CH1 is substituted with V185.

37. The antibody or fragment thereof according to any one of claims 33 to 35, wherein the HC1 CH1 substitution in F170 (EU numbering) is an F170I or F170V substitution.

38. The antibody or fragment thereof according to any one of claims 33 to 36, wherein the HC1 CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution.

39. The antibody or fragment thereof according to any one of claims 33 to 37, wherein the HC1 CH1 substitution in V185 (EU numbering) is a V185L substitution.

40. The antibody or fragment thereof according to any one of claims 33 to 38, wherein the LC1 CL1 substitution in L135 (EU numbering) is an L135F substitution.

41. The antibody or fragment thereof according to claim 33, wherein the HC1 CH1 includes or consists of the F170V, S183I and V185L (EU numbering) substitutions, and the LC1 CL includes or consists of the L135F (EU numbering) substitution.

42. The antibody or fragment thereof according to claim 33, wherein the HC1 CH1 comprises or consists of the F170I, S183L, and V185L (EU numbering) substitutions, and the LC1 CL comprises or consists of the L135F (EU numbering) substitution.

43. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), Each HC1 and HC2 comprises the heavy chain variable domain (VH) and the first heavy chain constant domain (CH1) of human IgG, and each LC1 and LC2 comprises the light chain variable domain (VL) and the light chain constant domain (CL) of human IgG. The HC1 CH1 includes or consists of substitutions for F126, F176, S189, V191, and C220 (EU numbering), and the LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering). The HC1 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), and the LC1 CL is an antibody or its fragment that does not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

44. The antibody or fragment thereof according to claim 43, wherein the HC CH1 substitution in F126 (EU numbering) is an F126C substitution.

45. The antibody or fragment thereof according to any one of claims 43 to 44, wherein the HC CH1 substitution in F170 (EU numbering) is an F170V or F170I substitution.

46. The antibody or fragment thereof according to any one of claims 43 to 45, wherein the HC CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution.

47. The antibody or fragment thereof according to any one of claims 43 to 46, wherein the HC CH1 substitution in V185 (EU numbering) is a V185L substitution.

48. The antibody or fragment thereof according to any one of claims 43 to 47, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution.

49. The antibody or fragment thereof according to any one of claims 43 to 48, wherein the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC CL substitution at position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC CL substitution at position 124 (EU numbering) is a Q124C (kappa) substitution.

50. The antibody or fragment thereof according to any one of claims 43 to 49, wherein the LC CL substitution in L135 (EU numbering) is an L135F substitution.

51. The antibody or fragment thereof according to any one of claims 43 to 50, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution.

52. The antibody or fragment thereof according to claim 43, wherein the HC CH1 comprises or consists of the substitutions F126C, F170V, S183I, V185L, and C220S (EU numbering), and the LC CL comprises or consists of the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally comprising or consisting of the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally comprising or consisting of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering).

53. The antibody or fragment thereof according to claim 43, wherein the HC CH1 comprises or consists of the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and the LC CL comprises or consists of the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally comprising or consisting of the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally comprising or consisting of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering).

54. An antibody or a fragment thereof, First heavy chain (HC1), second heavy chain (HC2), first light chain (LC1), Each HC1 and HC2 comprises the heavy chain variable domain (VH) and the first heavy chain constant domain (CH1) of human IgG, and each LC1 and LC2 comprises the light chain variable domain (VL) and the light chain constant domain (CL) of human IgG. The HC1 CH1 includes or consists of substitutions for F126 and C220 (EU numbering), the LC1 CL includes or consists of substitutions for 124 (E124 for lambda, Q124 for kappa) and C214 (EU numbering), the HC2 CH1 includes or consists of substitutions for F170, S183, and V185 (EU numbering), the LC2 CL includes or consists of substitution L135 (EU numbering), and the HC1 CH1 and / or the HC2 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), The LC1 CL and / or the LC2 CL are antibodies or fragments thereof that do not contain any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

55. The antibody according to claim 54, wherein the HC1 CH1 substitution at F126 (EU numbering) is an F126C substitution, the LC1 CL substitution at position 124 (EU numbering) is an E124C substitution for lambda or a Q124C substitution for kappa LC, and optionally, the LC1 CL substitution at position 124 (EU numbering) is an E124C substitution for lambda, or optionally, the LC1 CL substitution at position 124 (EU numbering) is a Q124C substitution for kappa LC.

56. The antibody according to any one of claims 54 to 55, wherein the HC CH1 substitution in C220 (EU numbering) is a C220S substitution.

57. The antibody according to any one of claims 54 to 56, wherein the LC CL substitution in C214 (EU numbering) is a C214S substitution.

58. The antibody or fragment thereof according to any one of claims 26 to 57, wherein the antibody comprises two heavy / light chain pairs, the first pair being HC1 / LC1 and the second pair being HC2 / LC2.

59. The antibody according to any one of claims 26 to 57, further comprising a pair of HC1 and HC2.

60. The antibody according to claim 59, wherein HC1 and HC2 are paired via charge-charge interaction.

61. The antibody according to any one of claims 26 to 60, wherein each HC1 and HC2 further comprises a heavy chain constant domain (CH3), and the CH3 domain of HC1 and the CH3 domain of HC2 together constitute a knob-into-hole construct.

62. The antibody according to claim 61, wherein the HC1 CH3 constitutes a knob and the HC2 CH3 constitutes a hole.

63. The antibody according to claim 61, wherein the HC1 CH3 constitutes a hole and the HC2 CH3 constitutes a knob.

64. The antibody according to any one of claims 62 to 63, wherein the HC1 CH3 and / or the HC2 CH3 comprises or consists of T366, L368, and Y407 (EU numbering) substitutions.

65. The antibody according to claim 64, wherein the HC1 CH3 and / or HC2 CH3 substitution in T366 (EU numbering) is a T366W or T366S substitution.

66. The antibody according to any one of claims 64 to 65, wherein the HC1 CH3 and / or HC2 CH3 substitution in L368 (EU numbering) is an L368A substitution.

67. The antibody according to any one of claims 64 to 66, wherein the HC1 CH3 and / or HC2 CH3 substitution in Y407 (EU numbering) is a Y407V substitution.

68. The antibody according to any one of claims 64 to 67, wherein the HC1 CH3 contains or consists of the T366W (EU numbering) substitution, and the HC2 CH3 contains or consists of the T366S, L368A, and Y407V (EU numbering) substitutions.

69. The antibody according to any one of claims 64 to 68, wherein the HC1 CH3 comprises or consists of the T366S, L368A, and Y407V (EU numbering) substitutions, and the HC2 CH3 comprises or consists of the T366W (EU numbering) substitution.

70. The antibody according to any one of claims 26 to 69, wherein LC1 and / or LC2 are kappa light chains.

71. The antibody according to any one of claims 26 to 69, wherein LC1 and / or LC2 are lambda light chains.

72. The antibody according to any one of claims 26 to 69, wherein LC1 is a kappa light chain and LC2 is a lambda light chain.

73. The antibody according to any one of claims 26 to 69, wherein LC1 is a lambda light chain and LC2 is a kappa light chain.

74. The antibody or fragment thereof according to any one of claims 26 to 73, wherein the HC2 CH1 includes or consists of SEQ ID NO: 9, and the LC2 CL includes or consists of SEQ ID NO: 1 or 6.

75. The antibody according to any one of claims 26 to 74, wherein the light chain (LC) variable domain (VL) framework region (FR) and / or the heavy chain (HC) variable domain (VH) framework region (FR) comprises one or more substitutions.

76. The antibody according to claim 75, wherein the VL FR comprises substitution with Q38 and / or G100 and / or G101 (EU numbering).

77. The antibody according to claim 75, wherein the VH FR comprises substitution with Q39 and / or G44 (EU numbering).

78. The antibody according to any one of claims 75 to 77, wherein the VH FR includes substitution with Q39 and / or G44 (EU numbering), and the VL FR includes substitution with Q38 and / or G100 and / or G101 (EU numbering).

79. The antibody according to any one of claims 76 to 78, wherein the VL FR substitution in Q38 includes, or consists of, a substitution selected from the group consisting of Q38D and Q38K substitutions (EU numbering).

80. The antibody according to any one of claims 76 to 79, wherein the VL FR substitution in G100 includes or consists of a substitution selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering).

81. The antibody according to any one of claims 76 to 80, wherein the VL FR substitution in G101 includes or consists of a substitution selected from the group consisting of G101D and G101K substitutions (EU numbering).

82. The antibody according to any one of claims 77 to 81, wherein the VH FR substitution in Q39 includes, or consists of, a substitution selected from the group consisting of Q39D and Q39K substitutions (EU numbering).

83. The antibody according to any one of claims 77 to 82, wherein the VH FR substitution in G44 includes or consists of a substitution selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering).

84. The antibody according to any one of claims 75 to 83, wherein the VH FR substitution includes or consists of a Q39K substitution, and the VL FR substitution includes or consists of a Q38D substitution.

85. The antibody according to any one of claims 75 to 83, wherein the VH FR substitution includes or consists of a Q39D substitution, and the VL FR substitution includes or consists of a Q38K substitution.

86. The antibody according to any one of claims 75 to 85, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G100D substitution.

87. The antibody according to any one of claims 75 to 85, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G100K substitution.

88. The antibody according to any one of claims 75 to 85, wherein the substitution of VH FR includes or consists of a G44C substitution, and the substitution of VL FR includes or consists of a G100C substitution.

89. The antibody according to any one of claims 75 to 85, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G101D substitution.

90. The antibody according to any one of claims 75 to 85, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G101K substitution.

91. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a Q39K substitution, the substitution of LC1 VL FR includes or consists of a Q38D substitution, the substitution of HC2 VH FR includes or consists of a Q39D substitution, and the substitution of LC2 VL FR includes or consists of a Q38K substitution.

92. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a Q39D substitution, the substitution of LC1 VL FR includes or consists of a Q38K substitution, the substitution of HC2 VH FR includes or consists of a Q39K substitution, and the substitution of LC2 VL FR includes or consists of a Q38D substitution.

93. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G100D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G100K substitution.

94. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G100K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G100D substitution.

95. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution.

96. The antibody according to any one of claims 75 to 90, wherein the substitution of HC2 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution.

97. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G101D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G101K substitution.

98. The antibody according to any one of claims 75 to 90, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G101K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G101D substitution.

99. The antibody according to any one of claims 75 to 98, wherein the HC1 or HC2 VH comprises or consists of one of sequence numbers 53 to 64.

100. The antibody according to any one of claims 75 to 99, wherein the LC1 or the LC2 VL contains or consists of one of the sequence numbers 65 to 80.

101. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 30, 43, 35, and 52, or Sequence IDs 35, 52, 30, and 43 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

102. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 29, 44, 36, and 51, or Sequence IDs 36, 51, 29, and 44 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

103. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 48, or Sequence IDs 33, 48, 28, and 39 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

104. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 47, or Sequence IDs 34, 47, 27, and 40 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

105. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 26, 38, 31, and 45, or Sequence IDs 31, 45, 26, and 38 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

106. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 25, 37, 32, and 46, or Sequence IDs 32, 46, 25, and 37 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

107. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 28, 41, 33, and 50, or Sequence IDs 33, 50, 28, and 41 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

108. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 27, 42, 34, and 49, or Sequence IDs 34, 49, 27, and 42 An antibody according to any one of claims 75 to 100, comprising or consisting of the sequence of .

109. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 30, LC1 contains or comprises SEQ ID NO: 43, HC2 contains or comprises SEQ ID NO: 35, and LC2 contains or comprises SEQ ID NO:

52.

110. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 29, LC1 contains or comprises SEQ ID NO: 44, HC2 contains or comprises SEQ ID NO: 36, and LC2 contains or comprises SEQ ID NO:

51.

111. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 28, LC1 contains or comprises SEQ ID NO: 39, HC2 contains or comprises SEQ ID NO: 33, and LC2 contains or comprises SEQ ID NO:

48.

112. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 27, LC1 contains or comprises SEQ ID NO: 40, HC2 contains or comprises SEQ ID NO: 34, and LC2 contains or comprises SEQ ID NO:

47.

113. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 26, LC1 contains or comprises SEQ ID NO: 38, HC2 contains or comprises SEQ ID NO: 31, and LC2 contains or comprises SEQ ID NO:

45.

114. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 25, LC1 contains or comprises SEQ ID NO: 37, HC2 contains or comprises SEQ ID NO: 32, and LC2 contains or comprises SEQ ID NO:

46.

115. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 28, LC1 contains or comprises SEQ ID NO: 41, HC2 contains or comprises SEQ ID NO: 33, and LC2 contains or comprises SEQ ID NO:

50.

116. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 27, LC1 contains or comprises SEQ ID NO: 42, HC2 contains or comprises SEQ ID NO: 34, and LC2 contains or comprises SEQ ID NO:

49.

117. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 35, LC1 contains or comprises SEQ ID NO: 52, HC2 contains or comprises SEQ ID NO: 30, and LC2 contains or comprises SEQ ID NO:

43.

118. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 36, LC1 contains or comprises SEQ ID NO: 51, HC2 contains or comprises SEQ ID NO: 29, and LC2 contains or comprises SEQ ID NO:

44.

119. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 33, LC1 contains or comprises SEQ ID NO: 48, HC2 contains or comprises SEQ ID NO: 28, and LC2 contains or comprises SEQ ID NO:

39.

120. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 34, LC1 contains or comprises SEQ ID NO: 47, HC2 contains or comprises SEQ ID NO: 27, and LC2 contains or comprises SEQ ID NO:

40.

121. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 31, LC1 contains or comprises SEQ ID NO: 45, HC2 contains or comprises SEQ ID NO: 26, and LC2 contains or comprises SEQ ID NO:

38.

122. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 32, LC1 contains or comprises SEQ ID NO: 46, HC2 contains or comprises SEQ ID NO: 25, and LC2 contains or comprises SEQ ID NO:

37.

123. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 33, LC1 contains or comprises SEQ ID NO: 50, HC2 contains or comprises SEQ ID NO: 28, and LC2 contains or comprises SEQ ID NO:

41.

124. The antibody according to any one of claims 101 to 108, wherein HC1 contains or comprises SEQ ID NO: 34, LC1 contains or comprises SEQ ID NO: 49, HC2 contains or comprises SEQ ID NO: 27, and LC2 contains or comprises SEQ ID NO:

42.

125. A composition comprising one or more polynucleotides encoding a Fab and / or antibody according to any one of claims 1 to 124.

126. The composition according to claim 125, further comprising one or more polynucleotides encoding an IgG Fab or antibody.

127. An expression vector or construct for the expression of one or more polynucleotides according to any one of claims 125 to 126.

128. The expression vector or construct according to claim 127, wherein the expression vector is a plasmid or a viral vector.

129. A cell comprising one or more polynucleotides, expression vectors, or constructs according to any one of claims 125 to 128.

130. A cell expressing one or more polynucleotides encoding a Fab and / or antibody or a fragment thereof, as described in any one of claims 1 to 124.

131. The aforementioned cells are mammalian cells A cell according to any one of claims 129 to 130, which is a bacterial cell.

132. The cell according to any one of claims 129 to 131, wherein the cell is a HEK293 cell, a Chinese hamster ovary (CHO) cell, or an NS0 cell.

133. A pharmaceutical composition comprising a Fab and / or antibody or a fragment thereof according to any one of claims 1 to 124.

134. A composition comprising the cells described in any one of claims 129 to 132, wherein the composition is optionally a cell culture.

135. A composition comprising the cell supernatant and / or cell lysate of the cells according to any one of claims 129 to 132.

136. A composition comprising an antibody comprising two heavy / light chain pairs selected from the pairs HC1 / LC1, HC2 / LC2, HC1 / LC2, and HC2 / LC1 described in any one of claims 1 to 124, wherein at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibody comprises the HC1 / LC1 pair and the HC2 / LC2 pair.

137. The composition according to claim 136, wherein the composition does not contain an antibody comprising either HC1 / LC2 or HC2 / LC1, or contains 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less of the antibody.

138. The composition according to any one of claims 133 to 137, wherein the composition is a cell culture supernatant and / or cell lysate, and the composition contains at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibody comprising the HC1 / LC1 pair and the HC2 / LC2 pair before any concentration of the composition with respect to the antibody comprising the HC1 / LC1 pair and the HC2 / LC2 pair.

139. The composition according to claim 138, wherein the composition does not contain an antibody or fragment thereof containing either the HC1 / LC2 pair or the HC2 / LC1 pair, or contains 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less of such antibody before any concentration of the composition with respect to the antibody containing the HC1 / LC1 pair and the HC2 / LC2 pair.

140. A method for producing an antibody or a fragment thereof, wherein the method is The present invention provides a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL). The HC1 CH1 includes substitution with F170 and S183, and substitution with V185 (EU numbering), and the LC1 CL includes substitution with L135 (EU numbering), The aforementioned HC1 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), A method in which the LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

141. The method according to claim 140, wherein the HC1 CH1 consists of substitutions in F170 and S183 (EU numbering), or F170, S183 and V185 (EU numbering), and the LC1 CL consists of substitutions in L135 (EU numbering).

142. A method for producing an antibody or fragment thereof according to claim 141, further comprising substitution of HC1 CH1 with V185.

143. A method for producing an antibody or fragment thereof according to any one of claims 140 to 142, wherein the HC1 CH1 substitution in F170 (EU numbering) is F170I or F170V substitution.

144. A method for producing an antibody or fragment thereof according to any one of claims 140 to 143, wherein the HC1 CH1 substitution in S183 (EU numbering) is an S183L or S183I substitution.

145. A method for producing an antibody or fragment thereof according to any one of claims 140 to 144, wherein the HC1 CH1 substitution in V185 (EU numbering) is a V185L substitution.

146. A method for producing an antibody or fragment thereof according to any one of claims 140 to 145, wherein the LC1 CL1 substitution in L135 (EU numbering) is an L135F substitution.

147. A method for producing an antibody or fragment thereof according to any one of claims 140 to 146, wherein the HC1 CH1 consists of F170I and S183V (EU numbering) substitutions, and the LC1 CL consists of L135F (EU numbering) substitution.

148. A method for producing an antibody or fragment thereof according to any one of claims 140 to 147, wherein the HC1 CH1 consists of F170I, S183L, and V185L (EU numbering) substitutions, and the LC1 CL consists of L135F (EU numbering) substitution.

149. A method for producing an antibody or a fragment thereof, wherein the method is The invention includes providing a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, Each HC1 and HC2 contains the heavy chain variable domain (VH) and the first heavy chain constant domain (CH1) of human IgG. Each LC1 and LC2 contains the variable light chain domain (VL) and the constant light chain domain (CL) of human IgG. The HC1 CH1 includes or consists of substitutions for F126, F170, S183, V185, and C220 (EU numbering). The LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) or Q124, L135, and C214 (kappa) (EU numbering), and optionally the LC1 CL includes or consists of substitutions for E124, L135, and C214 (lambda) (EU numbering), or optionally the LC1 CL includes or consists of substitutions for Q124, L135, and C214 (kappa) (EU numbering), The aforementioned HC1 CH1 does not include any substitution selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), A method in which the LC1 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

150. The method according to claim 149, wherein the HC1 CH1 substitution in F126 (EU numbering) is F126C substitution.

151. The method according to any one of claims 149 to 150, wherein the HC1 CH1 substitution in F170 (EU numbering) is F170V or F170I substitution.

152. The method according to any one of claims 149 to 151, wherein the HC1 CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.

153. The method according to any one of claims 149 to 152, wherein the HC1 CH1 substitution in V185 (EU numbering) is V185L substitution.

154. The method according to any one of claims 149 to 153, wherein the HC1 CH1 substitution in C220 (EU numbering) is a C220S substitution.

155. The method according to any one of claims 149 to 154, wherein the LC1 CL substitution at position 124 (EU numbering) is an E124C substitution for lambda or a Q124C substitution for kappa LC, and optionally, the LC1 CL substitution at position 124 (EU numbering) is an E124C substitution for lambda LC, or optionally, the LC1 CL substitution at position 124 (EU numbering) is a Q124C substitution for kappa LC.

156. The method according to claims 149 to 155, wherein the LC1 CL substitution in L135 (EU numbering) is an L135F substitution.

157. The method according to claims 149 to 156, wherein the LC1 CL substitution in C214 (EU numbering) is a C214S substitution.

158. The method according to claim 149, wherein HC1 CH1 includes or consists of the substitutions F126C, F170V, S183I, V185L, and C220S (EU numbering), and LC1 CL includes or consists of the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally, LC1 CL includes or consists of the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering).

159. The method according to claim 149, wherein HC1 CH1 includes or consists of the substitutions F126C, F170I, S183L, V185L, and C220S (EU numbering), and LC1 CL includes or consists of the substitutions E124C, L135F, and C214S (lambda), or Q124C, L135F, and C214S (kappa) (EU numbering), optionally, LC1 CL includes or consists of the substitutions E124C, L135F, and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitutions Q124C, L135F, and C214S (kappa) (EU numbering).

160. A method for producing an antibody or a fragment thereof, wherein the method is The present invention provides a first heavy chain (HC1), a second heavy chain (HC2), a first light chain (LC1), and a second (LC2) light chain, each HC1 and HC2 comprising a human IgG heavy chain variable domain (VH) and a first heavy chain constant domain (CH1), and each LC1 and LC2 comprising a human IgG light chain variable domain (VL) and a light chain constant domain (CL). The HC1 CH1 includes or consists of substitutions for F126 and C220 (EU numbering), the LC1 CL includes or consists of substitutions for E124 and C214 (lambda) or Q124 and C214 (kappa) (EU numbering) (optionally to E124 and C214 (lambda), or optionally to Q124 and C214 (kappa)), the HC2 CH1 includes or consists of substitutions for F170, S183, and V185 (EU numbering), the LC2 CL includes or consists of substitution L135 (EU numbering), The HC1 CH1 and / or the HC2 CH1 does not include any substitutions selected from L128F, A141M, A141T, A141I, F170M, F170Y, F170S, F170A, S181I, S181T, S181M, S183A, S183V, and V185A (EU numbering), A method in which the LC1 CL and / or the LC2 CL does not include any substitutions selected from F116A, F118V, S131T, S131D, V133A, V133I, L135Y, L135V, S162A, S162M, T164S, S174A, S176M, S176A, S176T, S176F, T178L, T178V, and T178I (EU numbering).

161. The method according to claim 160, wherein the HC1 CH1 substitution in F126 (EU numbering) is an F126C substitution, the LC1 CL substitution in position 124 (EU numbering) is an E124C (lambda) or Q124C (kappa) substitution, and optionally, the LC1 CL substitution in position 124 (EU numbering) is an E124C (lambda) substitution, or optionally, the LC1 CL substitution in position 124 (EU numbering) is a Q124C (kappa) substitution.

162. The method according to any one of claims 160 to 161, wherein the HC1 CH1 substitution in C220 (EU numbering) is a C220S substitution.

163. The antibody according to any one of claims 160 to 162, wherein the LC1 CL substitution in C214 (EU numbering) is a C214S substitution.

164. The method according to any one of claims 160 to 163, wherein the HC2 CH1 substitution in F170 (EU numbering) is F170V or F170I substitution.

165. The method according to any one of claims 160 to 164, wherein the HC2 CH1 substitution in S183 (EU numbering) is S183L or S183I substitution.

166. The method according to any one of claims 160 to 165, wherein the HC2 CH1 substitution in V185 (EU numbering) is V185L substitution.

167. The method according to any one of claims 160 to 166, wherein the LC2 CL substitution in L135 (EU numbering) is an L135F substitution.

168. The method according to claim 160, wherein HC1 CH1 includes or consists of the substitution of F126C and C220S (EU numbering), LC1 CL includes or consists of the substitution of E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) (optionally, LC1 CL includes or consists of the substitution of E124C and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitution of Q124C and C214S (kappa) (EU numbering)), HC2 CH1 includes or consists of F170I, S183L, V185L (EU numbering), and LC2 CL includes or consists of the substitution of L135F (EU numbering).

169. The method according to claim 160, wherein HC1 CH1 includes or consists of the substitution of F126C and C220S (EU numbering), LC1 CL includes or consists of the substitution of E124C and C214S (lambda) or Q124C and C214S (kappa) (EU numbering) (optionally, LC1 CL includes or consists of the substitution of E124C and C214S (lambda) (EU numbering), or optionally, LC1 CL includes or consists of the substitution of Q124C and C214S (kappa) (EU numbering)), HC2 CH1 includes or consists of F170V, S183I, V185L (EU numbering), and LC2 CL includes or consists of the substitution of L135F (EU numbering).

170. The method according to any one of claims 140 to 169, wherein an antibody comprising two heavy / light chain pairs is produced, the first pair being HC1 / LC1 and the second pair being HC2 / LC2.

171. The method according to any one of claims 140 to 170, wherein the antibody further comprises an HC1 / HC2 pair.

172. The method according to claim 171, wherein HC1 and HC2 are paired via charge-charge interaction.

173. The method according to any one of claims 140 to 172, wherein each HC1 and HC2 further comprises a heavy chain constant domain (CH3), and the HC1 CH3 and the HC2 CH3 together constitute a knob-into-hole structure.

174. The method according to claim 173, wherein the HC1 CH3 and / or the HC2 CH3 constitute a knob, and the HC2 CH3 constitutes a hole.

175. The method according to claim 173, wherein the HC1 CH3 constitutes a hole and the HC2 CH3 constitutes a knob.

176. The method according to any one of claims 171 to 175, wherein the HC1 CH3 and / or the HC2 CH3 includes or consists of T366, L368, and Y407 (EU numbering) substitutions.

177. The method according to any one of claims 171 to 176, wherein the substitution of HC1 CH3 and / or HC2 CH3 in T366 (EU numbering) is a substitution of T366W or T366S.

178. The method according to any one of claims 171 to 177, wherein the substitution of HC1 CH3 and / or HC2 CH3 in L368 (EU numbering) is an L368A substitution.

179. The method according to any one of claims 171 to 178, wherein the substitution of HC1 CH3 and / or HC2 CH3 in Y407 (EU numbering) is a substitution of Y407V.

180. The method according to any one of claims 171 to 179, wherein the HC1 CH3 includes or consists of the T366W (EU numbering) substitution, and the HC2 CH3 includes or consists of the T366S, L368A, and Y407V (EU numbering) substitutions.

181. The method according to any one of claims 171 to 180, wherein the HC1 CH3 includes or consists of the T366S, L368A, and Y407V (EU numbering) substitutions, and the HC2 CH3 includes or consists of the T366W (EU numbering) substitution.

182. The method according to any one of claims 140 to 181, wherein LC1 and / or LC2 are kappa light chains.

183. The method according to any one of claims 140 to 181, wherein LC1 and / or LC2 are lambda light chains.

184. The method according to any one of claims 140 to 181, wherein LC1 is a lambda light chain and LC2 is a kappa light chain.

185. The method according to any one of claims 140 to 181, wherein LC1 is a kappa light chain and LC2 is a lambda light chain.

186. The method according to any one of claims 140 to 185, wherein the VL FR is substituted with Q38, and / or G100, and / or G101 (EU numbering).

187. The method according to any one of claims 140 to 186, wherein the VH FR includes substitution with Q39 and / or G44 (EU numbering).

188. The method according to any one of claims 140 to 187, wherein the VH FR includes substitution for Q39 and / or G44 (EU numbering), and the VL FR includes substitution for Q38 and / or G100 and / or G101 (EU numbering).

189. The method according to any one of claims 140 to 188, wherein the VL FR substitution in Q38 includes, or consists of, a substitution selected from the group consisting of Q38D and Q38K substitutions (EU numbering).

190. The method according to any one of claims 140 to 188, wherein the VL FR substitution in G100 includes, or consists of, a substitution selected from the group consisting of G100D, G100K, and G100C substitutions (EU numbering).

191. The method according to any one of claims 140 to 188, wherein the VL FR substitution in G101 includes, or consists of, a substitution selected from the group consisting of G101D and G101K substitutions (EU numbering).

192. The method according to any one of claims 140 to 188, wherein the VH FR substitution in Q39 includes, or consists of, a substitution selected from the group consisting of Q39D and Q39K substitutions (EU numbering).

193. The method according to any one of claims 140 to 188, wherein the VH FR substitution in G44 includes, or consists of, substitutions selected from the group consisting of G44K, G44D, and G44C substitutions (EU numbering).

194. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a Q39K substitution, and the substitution of VL FR includes or consists of a Q38D substitution.

195. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a Q39D substitution, and the substitution of VL FR includes or consists of a Q38K substitution.

196. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G100D substitution.

197. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G100K substitution.

198. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a G44C substitution, and the substitution of VL FR includes or consists of a G100C substitution.

199. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a G44K substitution, and the substitution of VL FR includes or consists of a G101D substitution.

200. The method according to any one of claims 140 to 193, wherein the substitution of VH FR includes or consists of a G44D substitution, and the substitution of VL FR includes or consists of a G101K substitution.

201. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a Q39K substitution, the substitution of LC1 VL FR includes or consists of a Q38D substitution, the substitution of HC2 VH FR includes or consists of a Q39D substitution, and the substitution of LC2 VL FR includes or consists of a Q38K substitution.

202. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a Q39D substitution, the substitution of LC1 VL FR includes or consists of a Q38K substitution, the substitution of HC2 VH FR includes or consists of a Q39K substitution, and the substitution of LC2 VL FR includes or consists of a Q38D substitution.

203. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G100D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G100K substitution.

204. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G100K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G100D substitution.

205. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution.

206. The method according to any one of claims 140 to 193, wherein the substitution of HC2 VH FR includes or consists of a G44C substitution, and the substitution of LC1 VL FR includes or consists of a G100C substitution.

207. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44K substitution, the substitution of LC1 VL FR includes or consists of a G101D substitution, the substitution of HC2 VH FR includes or consists of a G44D substitution, and the substitution of LC2 VL FR includes or consists of a G101K substitution.

208. The method according to any one of claims 140 to 193, wherein the substitution of HC1 VH FR includes or consists of a G44D substitution, the substitution of LC1 VL FR includes or consists of a G101K substitution, the substitution of HC2 VH FR includes or consists of a G44K substitution, and the substitution of LC2 VL FR includes or consists of a G101D substitution.

209. The method according to any one of claims 140 to 208, wherein the HC1 or the HC2 VH includes or consists of any one of sequence numbers 53 to 64.

210. The method according to any one of claims 140 to 209, wherein LC1 or LC2 VL includes or consists of one of sequence numbers 65 to 80.

211. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 30, 43, 35, and 52, or Sequence IDs 35, 52, 30, and 43 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

212. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 29, 44, 36, and 51, or Sequence IDs 36, 51, 29, and 44 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

213. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 48, or Sequence IDs 33, 48, 28, and 39 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

214. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 47, or Sequence IDs 34, 47, 27, and 40 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

215. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 26, 38, 31, and 45, or Sequence IDs 31, 45, 26, and 38 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

216. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 25, 37, 32, and 46, or Sequence IDs 32, 46, 25, and 37 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

217. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 28, 39, 33, and 50, or Sequence IDs 33, 50, 28, and 41 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

218. Each of the heavy chain and light chain of the HC1 / LC1 pair and each of the heavy chain and light chain of the HC2 / LC2 pair are, Sequence IDs 27, 40, 34, and 49, or Sequence IDs 34, 49, 27, and 42 An antibody according to any one of claims 140 to 210, comprising or consisting of the sequence of .

219. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 30, LC1 includes or consists of sequence number 43, HC2 includes or consists of sequence number 35, and LC2 includes or consists of sequence number 52.

220. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 29, LC1 includes or consists of sequence number 44, HC2 includes or consists of sequence number 36, and LC2 includes or consists of sequence number 51.

221. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 39, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 48.

222. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 40, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 47.

223. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 26, LC1 includes or consists of sequence number 38, HC2 includes or consists of sequence number 31, and LC2 includes or consists of sequence number 45.

224. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 25, LC1 includes or consists of sequence number 37, HC2 includes or consists of sequence number 32, and LC2 includes or consists of sequence number 46.

225. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 28, LC1 includes or consists of sequence number 41, HC2 includes or consists of sequence number 33, and LC2 includes or consists of sequence number 50.

226. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 27, LC1 includes or consists of sequence number 42, HC2 includes or consists of sequence number 34, and LC2 includes or consists of sequence number 49.

227. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 35, LC1 includes or consists of sequence number 52, HC2 includes or consists of sequence number 30, and LC2 includes or consists of sequence number 43.

228. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 36, LC1 includes or consists of sequence number 51, HC2 includes or consists of sequence number 29, and LC2 includes or consists of sequence number 44.

229. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 48, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 39.

230. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 47, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 40.

231. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 31, LC1 includes or consists of sequence number 45, HC2 includes or consists of sequence number 26, and LC2 includes or consists of sequence number 38.

232. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 32, LC1 includes or consists of sequence number 46, HC2 includes or consists of sequence number 25, and LC2 includes or consists of sequence number 37.

233. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 33, LC1 includes or consists of sequence number 50, HC2 includes or consists of sequence number 28, and LC2 includes or consists of sequence number 41.

234. The method according to any one of claims 201 to 218, wherein HC1 includes or consists of sequence number 34, LC1 includes or consists of sequence number 49, HC2 includes or consists of sequence number 27, and LC2 includes or consists of sequence number 42.

235. The method according to any one of claims 140 to 234, wherein multiple antibodies comprising two heavy / light chain pairs are produced, and at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the antibodies comprising two heavy / light chain pairs produced comprises HC1 / LC1 and HC2 / LC2.

236. The method according to any one of claims 140 to 235, wherein 0% or 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or less of the antibody containing two heavy / light chain pairs produced contains either HC1 / LC2 or HC2 / LC1.

237. The method according to claim 236, wherein the percentage (%) of the antibody containing the HC1 / LC1 pair and the HC2 / LC2 pair refers to the percentage of the HC1 / LC1 pair and the HC2 / LC2 pair of the antibody containing the HC1 / LC1 pair and the HC2 / LC2 pair before any enrichment.

238. The method according to claim 236, wherein the percentage (%) of the antibody containing the HC1 / LC2 pair and the HC2 / LC1 pair refers to the percentage of the HC1 / LC2 pair and the HC2 / LC1 pair of the antibody containing the HC1 / LC1 pair and the HC2 / LC2 pair before any enrichment.

239. The method according to any one of claims 140 to 238, wherein the provision comprises providing one or more polynucleotides encoding HC1, HC2, LC1, and LC2.

240. The method according to claim 239, wherein one or more polynucleotides encode LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately the ratios thereof, and optionally the ratios thereof are 1:5 or approximately 1:

5.

241. The method according to claim 240, wherein the one or more polynucleotides encode HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately the ratios described above.

242. The method according to claim 241, wherein one or more polynucleotides encode HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately the ratios described above.

243. The method according to claim 239, wherein the one or more polynucleotides encode HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately the ratios described above.

244. The method according to any one of claims 239 to 243, wherein one or more polynucleotides encode an IgG Fab or an antibody.

245. The method according to any one of claims 141 to 239, wherein the provision includes providing one or more expression vectors or constructs encoding the HC1, the HC2, the LC1, and the LC2.

246. The method according to claim 245, wherein one or more expression vectors or constructs encode LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately the ratios thereof, and optionally the ratios thereof are 1:5 or approximately 1:

5.

247. The method according to claim 246, wherein the one or more expression vectors or constructs encode HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately the ratios described above.

248. The method according to claim 247, wherein one or more expression vectors or constructs encode HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately the ratios described above.

249. The method according to claim 245, wherein the one or more expression vectors or constructs encode HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately the ratios thereof.

250. The method according to any one of claims 245 to 249, wherein the one or more expression vectors are plasmids or viral vectors.

251. The method according to any one of claims 245 to 250, wherein the one or more expression vectors or constructs encode an IgG Fab or an antibody.

252. The method according to any one of claims 140 to 251, wherein the provision includes providing cells that express HC1, HC2, LC1, and LC2.

253. The method according to claim 252, wherein the cells express LC1 and LC2 in a ratio of 8:1 to 1:8, optionally 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, or approximately the ratios thereof, and optionally the ratio is 1:5 or approximately 1:

5.

254. The method according to claim 253, wherein the cells express HC1 and HC2 in a ratio of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8, or optionally in a ratio of 1:1, 1.5:1, or 2:1, or approximately the ratios described above.

255. The method according to claim 254, wherein the cells express HC1 and LC2 in a ratio of 1:0.5 to 1:3, optionally 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, or 1:3, or approximately the ratios described above.

256. The method according to claim 252, wherein the cells express HC1, HC2, LC1, and LC2 in a ratio of 1:1:1:1, 1:1:1.5:1.5, 1:1:2:1, or 2:2:5:1, or approximately the ratios thereof.

257. The method according to any one of claims 252 to 256, wherein the cells express an IgG Fab or an antibody.

258. The method according to any one of claims 252 to 257, wherein the cell is a mammalian cell or a bacterial cell.

259. The method according to any one of claims 252 to 257, wherein the cells are HEK293 cells, Chinese hamster ovary (CHO) cells, or NS0 cells.

260. The method according to any one of claims 140 to 259, further comprising extraction and / or purification of the antibody or a fragment thereof.

261. The method according to claim 260, wherein the antibody or its fragment is extracted and / or purified from the cell supernatant or cell lysate.

262. The Fab according to any one of claims 1 to 30, wherein the Fab is a chimeric Fab, a humanized Fab, or a human Fab.

263. The antibody, composition, cell, expression vector or construct, polynucleotide, or method according to any one of claims 31 to 261, wherein the antibody comprises or consists of an IgG antibody.

264. The antibody, composition, cell, expression vector or construct, polynucleotide, or method according to claim 263, wherein the IgG antibody is IgG1, IgG2, IgG3, or IgG4.

265. The antibody, composition, cell, expression vector or construct, polynucleotide, or method according to any one of claims 263 to 264, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

266. Any one of claims 1 to 265, wherein the fragment comprises an antigen-binding fragment.

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