DYNAMIC HUMAN HEAVY CHAIN ANTIBODY LIBRARIES

Abstract

Provided herein are libraries containing polynucleotides, where one of the polynucleotides encodes an antibody heavy chain with specific hypervariable regions HVR-H1 and HVR-H2. Further provided herein are libraries containing polynucleotides encoding a plurality of unique antibodies, wherein each antibody comprises a heavy chain variable region and a light chain variable region. Also provided are antibodies, polypeptide libraries, vector libraries, cells, non-human animals, antibody heavy chains, methods of making an antibody library, kits, and methods of generating a bispecific antibody related thereto.

Claims

1. A library comprising polynucleotides, wherein one of the polynucleotides encodes an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula I) X.sub.1TFX.sub.2X.sub.3YX.sub.4IHWV (SEQ ID NO:198), wherein X.sub.1 is F or Y, X.sub.2 is S or T, X.sub.3 is D, G, N, or S, and X.sub.4 is A, G, or W; (Formula II) YSIX.sub.1SGX.sub.2X.sub.3WX.sub.4WI (SEQ ID NO:199), wherein X.sub.1 is S or T, X.sub.2 is H or Y, X.sub.3 is H or Y, and X.sub.4 is A, D, G, N, S, or T; and (Formula III) FSLSTX.sub.1GVX.sub.2VX.sub.3WI (SEQ ID NO:200), wherein X.sub.1 is G or S, X.sub.2 is A or G, and X.sub.3 is A, G, S, or T; and wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula IV) LAX.sub.1IX.sub.2WX.sub.3X.sub.4DKX.sub.5YSX.sub.6SLKSRL (SEQ ID NO:201), wherein X.sub.1 is L or R, X.sub.2 is D or Y, X.sub.3 is A, D, S, or Y, X.sub.4 is D or G, X.sub.5 is R, S, or Y, and X.sub.6 is P or T; (Formula V) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV (SEQ ID NO:202), wherein X.sub.1 is A, D, E, S, or Y, X.sub.2 is S or Y, and X.sub.3 is H or Y; (Formula VI) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV (SEQ ID NO:203), wherein X.sub.1 is D, E, R, S, or Y, X.sub.2 is H or Y, X.sub.3 is N or S, and X.sub.4 is N or Y; (Formula VII) VSX.sub.1ISGX.sub.2GX.sub.3X.sub.4TYYADSVKGRF (SEQ ID NO:204), wherein X.sub.1 is A, G, S, V, or Y, X.sub.2 is A, D, S, or Y, X.sub.3 is D, G, or S, and X.sub.4 is S or T; (Formula VIII) IGX.sub.1INPNX.sub.2GX.sub.3TX.sub.4YAQKFQGRV (SEQ ID NO:205), wherein X.sub.1 is I, R, or W, X.sub.2 is F or R, X.sub.3 is D, G, or S, and X.sub.4 is K or N; (Formula IX) IGX.sub.1IX.sub.2PSX.sub.3GX.sub.4TX.sub.5YAQKFQGRV (SEQ ID NO:206), wherein X.sub.1 is I, R, or W, X.sub.2 is S or Y, X.sub.3 is G or S, X.sub.4 is D, G, or S, and X.sub.5 is K or N; and (Formula X) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTX.sub.5YAAX.sub.6VKGRF (SEQ ID NO:207), wherein X.sub.1 is K or R, X.sub.2 is A or T, X.sub.3 is D or Y, X.sub.4 is G or Y, X.sub.5 is D or E, and X.sub.6 is P or S.

2. The library of claim 1, wherein at least two, at least three, at least four, at least five or at least ten of the polynucleotides encode a heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula I) X.sub.1TFX.sub.2X.sub.3YX.sub.4IHWV (SEQ ID NO:198), wherein X.sub.1 is F or Y, X.sub.2 is S or T, X.sub.3 is D, G, N, or S, and X.sub.4 is A, G, or W; (Formula II) YSIX.sub.1SGX.sub.2X.sub.3WX.sub.4WI (SEQ ID NO:199), wherein X.sub.1 is S or T, X.sub.2 is H or Y, X.sub.3 is H or Y, and X.sub.4 is A, D, G, N, S, or T; and (Formula III) FSLSTX.sub.1GVX.sub.2VX.sub.3WI (SEQ ID NO:200), wherein X.sub.1 is G or S, X.sub.2 is A or G, and X.sub.3 is A, G, S, or T; and wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula IV) LAX.sub.1IX.sub.2WX.sub.3X.sub.4DKX.sub.5YSX.sub.6SLKSRL (SEQ ID NO:201), wherein X.sub.1 is L or R, X.sub.2 is D or Y, X.sub.3 is A, D, S, or Y, X.sub.4 is D or G, X.sub.5 is R, S, or Y, and X.sub.6 is P or T; (Formula V) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV (SEQ ID NO:202), wherein X.sub.1 is A, D, E, S, or Y, X.sub.2 is S or Y, and X.sub.3 is H or Y; (Formula VI) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV (SEQ ID NO:203), wherein X.sub.1 is D, E, R, S, or Y, X.sub.2 is H or Y, X.sub.3 is N or S, and X.sub.4 is N or Y; (Formula VII) VSX.sub.1ISGX.sub.2GX.sub.3X.sub.4TYYADSVKGRF (SEQ ID NO:204), wherein X.sub.1is A, G, S, V, or Y, X.sub.2 is A, D, S, or Y, X.sub.3 is D, G, or S, and X.sub.4 is S or T; (Formula VIII) IGX.sub.1INPNX.sub.2GX.sub.3TX.sub.4YAQKFQGRV (SEQ ID NO:205), wherein X.sub.1 is I, R, or W, X.sub.2 is F or R, X.sub.3 is D, G, or S, and X.sub.4 is K or N; (Formula IX) IGX.sub.1IX.sub.2PSX.sub.3GX.sub.4TX.sub.5YAQKFQGRV (SEQ ID NO:206), wherein X.sub.1 is I, R, or W, X.sub.2 is S or Y, X.sub.3 is G or S, X.sub.4 is D, G, or S, and X.sub.5 is K or N; and (Formula X) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTX.sub.5YAAX.sub.6VKGRF (SEQ ID NO:207), wherein X.sub.1 is K or R, X.sub.2 is A or T, X.sub.3 is D or Y, X.sub.4 is G or Y, X.sub.5 is D or E, and X.sub.6 is P or S.

3. The library of claim 1, wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula XI) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV (SEQ ID NO:208), wherein X.sub.1 is A, D, or E, X.sub.2 is S or Y, and X.sub.3 is H or Y; (Formula XII) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV (SEQ ID NO:209), wherein X.sub.1 is D, E, or S, X.sub.2 is H or Y, X.sub.3 is N or S, and X.sub.4 is N or Y; and (Formula XIII) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTEYAAX.sub.5VKGRF (SEQ ID NO:210), wherein X.sub.1 is K or R, X.sub.2 is A or T, X.sub.3 is D or Y, X.sub.4 is G or Y, X.sub.5 is P or S.

4. The library of claim 1, wherein each of the polynucleotides encode a heavy chain comprising a HVR-H1, a HVR-H2 and a HVR-H3, and wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158.

5. The library of claim 1, wherein each of the polynucleotides encode a heavy chain comprising a HVR-H1, a HVR-H2 and a HVR-H3, and wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52.

6. The library of claim 1, wherein each of the polynucleotides encode a heavy chain comprising a HVR-H1, a HVR-H2 and a HVR-H3, and wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164.

7. The library of claim 1, wherein each of the polynucleotides encode a heavy chain comprising a HVR-H1, a HVR-H2 and a HVR-H3, and wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

8. The library of claim 1, wherein the polynucleotides contain less than about 6.5*10.sup.4unique combinations of HVR-H1 and HVR-H2 sequences.

9. The library of claim 8, wherein the polynucleotides contain less than about 6700 unique combinations of HVR-H1 and HVR-H2 sequences.

10. The library of claim 9, wherein the polynucleotides contain about 6660 or less unique combinations of HVR-H1 and HVR-H2 sequences.

11. The library of claim 1, wherein the heavy chain variable region comprises a HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, and a HVR-H2 of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164.

12. The library of claim 1, wherein the heavy chain variable region comprises a HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, and a HVR-H2 of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

13. The library of claim 1, wherein the heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VIII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (V); and a HVR- H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VIII).

14. The library of claim 1, wherein the heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:122; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:154, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:161; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:145, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:128; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:61; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:153, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:67; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:100; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:123; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:126; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:129; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:124; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:130; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:150, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:132; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:12, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:149, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:117; and a HVR- H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:134.

15. The library of claim 1, wherein the heavy chain variable region comprises a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

16. The library of claim 1, wherein the heavy chain variable region comprises a FW-H1 comprising the amino acid sequence of SEQ ID NO:165, a FW-H2 comprising the amino acid sequence of SEQ ID NO:166, a FW-H3 comprising the amino acid sequence of SEQ ID NO:167, and/or a FW-H4 comprising the amino acid sequence of SEQ ID NO:168.

17. The library of claim 1, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195.

18. The library of claim 1, wherein the polynucleotides encode full-length antibody heavy chains.

19. The library of claim 1, further comprising polynucleotides that encode antibody light chain variable regions.

20. The library of claim 19, wherein the antibody light chain variable regions comprise a HVR-L1, a HVR-L2 and a HVR-L3, wherein the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264 and/or the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274.

21. The library of claim 19, wherein the polynucleotides that encode antibody light chain variable regions include at least one unique sequence.

22. The library of claim 19, wherein the polynucleotides that encode antibody light chain variable regions include at least about 280 unique sequences.

23. The library of claim 19, wherein the polynucleotides that encode antibody light chain variable regions include at least about 10.sup.5 unique sequences.

24. The library of claim 19, wherein the polynucleotides encode a plurality of unique antibodies, wherein the heavy chain variable region of each antibody of the plurality comprises an identical sequence.

25-40. (canceled)

41. The library of claim 1, wherein at least one of the HVR-H1 and HVR-H2 of the antibody heavy chain variable region adopts multiple conformations, as assayed by structural determination and/or computational modeling

42. The library of claim 1, wherein at least one of the polynucleotides encoding the antibody heavy chain variable region is in a vector.

43. The library of claim 42, wherein the vector is an expression vector.

44. The library of claim 42, wherein the vector is a display vector.

45. The library of claim 1, wherein at least one of the polynucleotides encoding the antibody heavy chain variable region is in a cell.

46. The library of claim 45, wherein the cell is a bacterial, yeast, or mammalian cell.

47-69. (canceled)

70. A method of preparing a library comprising providing and assembling the polynucleotide sequences of the library of claim 1.

71. A method of making an antibody library comprising the steps: (a) selecting one, two or three heavy chain HVRs comprising a sequence having multiple conformations; and (b) assembling polynucleotide sequences to produce a library of polynucleotides encoding a plurality of antibody heavy chain variable region sequences.

72.-76. (canceled)

77. A kit comprising the library of polynucleotides of claim 1.

78-80. (canceled)

81. An antibody heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula I) X.sub.1TFX.sub.2X.sub.3YX.sub.4IHWV (SEQ ID NO:198), wherein X.sub.1 is F or Y, X.sub.2 is S or T, X.sub.3 is D, G, N, or S, and X.sub.4 is A, G, or W; (Formula II) YSIX.sub.1SGX.sub.2X.sub.3WX.sub.4WI (SEQ ID NO:199), wherein X.sub.1 is S or T, X.sub.2 is H or Y, X.sub.3 is H or Y, and X.sub.4 is A, D, G, N, S, or T; and (Formula III) FSLSTX.sub.1GVX.sub.2VX.sub.3WI (SEQ ID NO:200), wherein X.sub.1 is G or S, X.sub.2 is A or G, and X.sub.3 is A, G, S, or T; and wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula IV) LAX.sub.1IX.sub.2WX.sub.3X.sub.4DKX.sub.5YSX.sub.6SLKSRL (SEQ ID NO:201), wherein X.sub.1 is L or R, X.sub.2 is D or Y, X.sub.3 is A, D, S, or Y, X.sub.4 is D or G, X.sub.5 is R, S, or Y, and X.sub.6 is P or T; (Formula V) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV (SEQ ID NO:202), wherein X.sub.1 is A, D, E, S, or Y, X.sub.2 is S or Y, and X.sub.3 is H or Y; (Formula VI) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV (SEQ ID NO:203), wherein X.sub.1 is D, E, R, S, or Y, X.sub.2 is H or Y, X.sub.3 is N or S, and X.sub.4 is N or Y; (Formula VII) VSX.sub.1ISGX.sub.2GX.sub.3X.sub.4TYYADSVKGRF (SEQ ID NO:204), wherein X.sub.1 is A, G, S, V, or Y, X.sub.2 is A, D, S, or Y, X.sub.3 is D, G, or S, and X.sub.4 is S or T; (Formula VIII) IGX.sub.1INPNX.sub.2GX.sub.3TX.sub.4YAQKFQGRV (SEQ ID NO:205), wherein X.sub.1 is I, R, or W, X.sub.2 is F or R, X.sub.3 is D, G, or S, and X.sub.4 is K or N; (Formula IX) IGX.sub.1IX.sub.2PSX.sub.3GX.sub.4TX.sub.5YAQKFQGRV (SEQ ID NO:206), wherein X.sub.1 is I, R, or W, X.sub.2 is S or Y, X.sub.3 is G or S, X.sub.4 is D, G, or S, and X.sub.5 is K or N; and (Formula X) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTX.sub.5YAAX.sub.6VKGRF (SEQ ID NO:207), wherein X.sub.1 is K or R, X.sub.2 is A or T, X.sub.3 is D or Y, X.sub.4 is G or Y, X.sub.5 is D or E, and X.sub.6 is P or S.

82. A non-human animal comprising polynucleotides, wherein one of the polynucleotides encodes the antibody heavy chain variable region of claim 81.

83. A phage comprising at least one polypeptide displayed on its surface, wherein the at least one polypeptide comprises the antibody heavy chain variable region of claim 81.

84. An antibody heavy chain comprising the antibody heavy chain variable region of claim 81.

85. . An antibody comprising a heavy chain and a light chain, wherein the heavy chain is the antibody heavy chain of claim 84.

86. A library comprising antigen binding domains, wherein one of the antigen binding domains comprises the antibody heavy chain variable region of claim 81.

87. A method of generating a bispecific antibody comprising two antibody heavy chain variable regions and two identical light chain variable regions, comprising: (a) screening for a first antigen binding domain that binds to a first antigen, wherein the first antigen binding domain comprises a first antibody heavy chain variable region and a first antibody light chain variable region, wherein the first antibody heavy chain variable region is the antibody heavy chain variable region of claim 81; (b) screening for a second antigen binding domain that binds to a second antigen, wherein the second antigen binding domain comprises a second antibody heavy chain variable region and a second antibody light chain variable region, wherein the second antibody heavy chain variable region has the same sequence as the first antibody heavy chain variable region; and (c) producing a bispecific antibody comprising the first antigen binding domain and the second antigen binding domain.

88. A bispecific antibody comprising: (a) a first binding domain comprising a first heavy chain variable region and a first light chain variable region, wherein the first binding domain binds to a first target; (b) a second binding domain comprising a second heavy chain variable region and a second light chain variable region, wherein the second binding domain binds to a second target, wherein the second heavy chain variable region has a sequence identical to the first heavy chain variable region sequence; and wherein the first antibody heavy chain variable region is the antibody heavy chain variable region of claim 81.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1A shows an entropy plot by residue number for the amino acids of a V.sub.H domain. 113 V.sub.H structures of human antibodies were used to calculate the entropy.

[0068] FIG. 1B shows the definition of the hyper-variable regions (HVRs) used herein for an exemplary antibody heavy chain variable domain (VH) sequence (SEQ ID NO:197) in comparison to the Kabat definition of the complementarity-determining regions (CDRs) for the same VII sequence.

[0069] FIG. 2A shows the affinity measurements for fabs with confirmed binding to the antigens TAGT-1 to TAGT-12.

[0070] FIG. 2B shows the melting temperature (Tm) measurements for fabs with confirmed binding to the antigens TAGT-1 to TAGT-12.

DETAILED DESCRIPTION

[0071] The present disclosure provides libraries containing synthetic (e.g., non-naturally occurring) polynucleotides that encode antibody heavy chains (e.g., heavy chains of a dynamic human antibody). Advantageously, the antibody heavy chains disclosed herein include HVR sequences designed to generate highly flexible loops for more effective substrate binding and/or specificity against multiple substrates of interest. These HVR sequences allow the creation of smaller antibody libraries with broader epitope coverage than existing techniques.

I. General Techniques

[0072] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 1993).

II. Definitions

[0073] Before describing the present disclosure in detail, it is to be understood that this present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0074] As used in this specification and the appended claims, the singular forms a, an and the include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a molecule optionally includes a combination of two or more such molecules, and the like.

[0075] The term about as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to about a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0076] It is understood that aspects and embodiments of the present disclosure described herein include comprising, consisting, and consisting essentially of aspects and embodiments.

[0077] The term antibody is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., a single-chain variable fragment or scFv) so long as they exhibit the desired biological activity.

[0078] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. The pairing of a V.sub.H and V.sub.L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

[0079] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa () and lambda (), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (), delta (), epsilon (), gamma () and mu (), respectively. The and classes are further divided into subclasses (isotypes) on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al., Cellular and Molecular Immunology, 4.sup.th ed. (W.B. Saunders Co., 2000).

[0080] The variable region or variable domain of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as V.sub.H. The variable domain of the light chain may be referred to as V.sub.L. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0081] The term variable domain residue numbering as in Kabat or amino acid position numbering as in Kabat, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a standard Kabat numbered sequence.

[0082] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The EU numbering system or EU index is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The EU index as in Kabat refers to the residue numbering of the human IgG1 EU antibody.

[0083] The term constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the C.sub.H1, C.sub.H2 and C.sub.H3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0084] The term full-length antibody (the terms intact antibody or whole antibody may be used interchangeably herein) may refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Similarly, the term full-length antibody heavy chain (the terms intact antibody heavy chain or whole antibody heavy chain may be used interchangeably herein) may refer to an antibody heavy chain in its substantially intact form, as opposed to an antibody heavy chain fragment. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0085] The term monoclonal antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA 101(34):12467-472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0086] As used herein, hypervariable region (HVR) refers to the regions of an antibody domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Each VH and VL is composed of three HVRs and four framework (FW) regions arranged from amino terminus to carboxy terminus in the following order: FW1-HVR1-FW2-HVR2-FW3-HVR3-FW4. Throughout the present disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Throughout the present disclosure, the four framework regions of the heavy chain are referred to as FW-H1, FW-H2, FW-H3 and FW-H4. For comparison, the definition of the HVRs (as used herein) is contrasted with the Kabat definition of the complementarity-determining regions (CDRs) (Yvonne Chen et al. (1999) Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Complex with Antigen, J. Mol. Biol. 293, 865-881) for the exemplary antibody heavy chain variable domain shown in FIG. 1B.

[0087] As used herein, library refers to a set of two or more entities having a shared class. For example, a library containing polynucleotides may refer to a set of two or more polynucleotides. The term library is used herein in the broadest sense and specifically covers sub-libraries that may or may not be combined.

[0088] As used herein, unique refers to a member of a set that is different from other members of the set. For example, a unique antibody from a library encoding a plurality of polynucleotides encoding antibodies may refer to an antibody having a particular sequence not shared by other antibodies encoded by the library. As a practical matter, it is to be understood that a unique member of a physical realization of a library may be present in more than one copy. For example, a library may contain a plurality of unique antibodies, with one or more of the unique antibody molecules occurring in more than one copy.

[0089] As used herein, diversity refers to a variety and/or heterogeneity. For example, a diversity of antibodies in a library may refer to a variety of antibodies with unique sequences present in the library.

[0090] The terms polypeptide, protein, and peptide are used interchangeably herein and may refer to polymers of two or more amino acids.

[0091] Polynucleotide, or nucleic acid, as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may comprise modification(s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, caps, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotides(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5 and 3 terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2-O-methyl-, 2-O-allyl-, 2-fluoro- or 2-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', CO, or CH2 (formacetal), in which each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (O) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0092] A cell (e.g., a cell or population of cells comprising a synthetic polynucleotide or library of synthetic polynucleotides) includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) (e.g., a synthetic polynucleotide that encodes an antibody heavy chain variable region of the present disclosure).

[0093] A non-human animal refers to any animal not classified as a human, such as domestic, farm, or zoo animals, sports, pet animals (such as dogs, horses, cats, cows, etc.), as well as animals used in research. Research animals may refer without limitation to nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In preferred embodiments, the animal is one that produces antibodies.

III. Antibody Libraries and Generation of Libraries

[0094] Certain aspects of the present disclosure relate to libraries of polynucleotides, e.g., that encode an antibody heavy chain variable region (V.sub.H) or light chain variable region (V.sub.L). A library of the present disclosure can contain one or more polynucleotides encoding a heavy chain variable region comprising a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are any of the HVR-H1s and/or HVR-H2s described herein.

[0095] In some embodiments, a library of the present disclosure contains a smaller number of unique heavy chain HVR sequences and/or unique V.sub.H sequences than typical antibody libraries. Advantageously, such libraries can provide sufficient diversity for the identification of antibodies binding one or more of a number of antigens of interest while also allowing for more efficient screening due to the reduced library size. In some embodiments, a library of the present disclosure includes or consists of polynucleotides containing less than about 10000, less than about 9000, less than about 8000, or less than about 7000 unique combinations of HVR-H1 and HVR-H2 sequences. In certain embodiments, a library of the present disclosure includes or consists of polynucleotides containing about 6600 or less unique combinations of HVR-H1 and HVR-H2 sequences.

[0096] In some embodiments, a library contains a plurality of polynucleotides, with at least one of the polynucleotides encoding an antibody heavy chain variable region of the present disclosure (e.g., comprising a HVR-H1 and HVR-H2 of the present disclosure).

[0097] In some embodiments, one or more of the polynucleotides encode an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200). In some embodiments, one or more of the polynucleotides encode an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207). In some embodiments, the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210). In some embodiments, one or more of the polynucleotides encode an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and an HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula W) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207). In some embodiments, one or more of the polynucleotides encode an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHW, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and an HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or display vector).

[0098] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 500, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, at least 4750, at least 5000, at least 5250, at least 5500, at least 5750, at least 6000, at least 6250, or at least 6500 of the polynucleotides encodes an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and/or an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula W) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207); and/or less than about 6.5*10.sup.4 (e.g., less than about 6.5*10.sup.4, less than about 5.5*10.sup.4, less than about 2.5*10.sup.4, less than about 1*10.sup.4, less than about 6700, less than about 6660, less than about 5000, less than about 2500, less than about 1000, less than about 690, less than about 500, less than about 100, less than about 50, etc.), less than or equal to 62272, less than or equal to 60928, less than or equal to 54656, or less than or equal to 6660 of the polynucleotides encodes an antibody heavy chain variable region comprising and HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or display vector).

[0099] In some embodiments, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 500, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, at least 4750, at least 5000, at least 5250, at least 5500, at least 5750, at least 6000, at least 6250, or at least 6500 of the polynucleotides encodes an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and/or an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210); and/or less than about 6.5*10.sup.4 (e.g., less than about 6.5*10.sup.4, less than about 5.5*10.sup.4, less than about 2.5*10.sup.4, less than about 1*10.sup.4, less than about 6700, less than about 6660, less than about 5000, less than about 2500, less than about 1000, less than about 690, less than about 500, less than about 100, less than about 50, etc.), less than or equal to 62272, less than or equal to 60928, less than or equal to 54656, or less than or equal to 6660 of the polynucleotides encodes an antibody heavy chain variable region comprising and HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W(SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and (Formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T(SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or display vector).

[0100] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207).

[0101] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210).

[0102] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:19); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207).

[0103] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210).

[0104] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula W) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T(SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T(SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N(SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N(SEQ ID NO:206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S(SEQ ID NO:207).

[0105] In some embodiments, the polynucleotides in the library encodes an antibody heavy chain variable region comprising an HVR-H1 comprising the amino acid sequence according to the formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y(SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S(SEQ ID NO:210).

[0106] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158. In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52.

[0107] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164. In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

[0108] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of Formula (I), Formula (II), and Formula (III), or the HVR-2 comprises an amino acid sequence selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), Formula (X), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 7, 8, 9, 11, 13, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 33, 34, 38, 40, 42, 43, 45, 47, 49, 50, and 51, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 60, 63, 65, 66, 67, 70, 82, 89, 93, 95, 105, 109, 110, 117, 121, 122, 123, 124, 128, 129, 130, 131, 132, and 134. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 14, 15, 30, 32, 35, 37, 39, 41, 44, 46, and 48, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 56, 59, 61, 62, 64, 68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 72, 81, 83, 86, 90, 91, 99, 100, 103, 106, 107, 108, 112, 113, 116, 118, 126, 135, and 136. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 57, 58, 80, 84, 85, 87, 88, 92, 94, 96, 97, 98, 101, 102, 104, 111, 114, 115, 119, 120, 125, 127 and 133.

[0109] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H3 is any HVR-H3 known in the art. In some embodiments, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0110] The heavy chain HVR sequences described herein may be included in any combination in a library of the present disclosure. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, and an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, and an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, and an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, and an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:223-256. In some embodiments, a heavy chain variable region comprises an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164 and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164, and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136, and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0111] In certain embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VIII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (V); and a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VIII). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XI); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XI); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XI). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (X); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (X); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VIII); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (X). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XIII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XIII); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XIII).

[0112] In certain embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:122; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:154, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:161; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:145, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:128; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:61; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:153, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:67; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:100; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:123; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:126; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:129; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:3 1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:124; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:130; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:150, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:132; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:12, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:149, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:117; and a HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:134. In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:26, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1 51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:34, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:5, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:17, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:152, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:94; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:48, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:58; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:163; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:160; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:92; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:97; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:164; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:137, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:54; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:3, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:127; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:85; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:109; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:120; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:140, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:131; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:141, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:142, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:159; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:143, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:144, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:146, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:147, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:133; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:148, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; and a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:118.

[0113] In some embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are listed in Table 1. In some embodiments, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOS: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence selected from SEQ ID NOS: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195.

[0114] In some embodiments, a heavy chain variable region further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4). In some embodiments, one, two, three, or four of the framework sequences is/are the following:

TABLE-US-00001 (SEQIDNO:165) FW-H1isEVQLVESGGGLVQPGGSLRLSCAASG (SEQIDNO:166) FW-H2isRQAPGKGLEW (SEQIDNO:167) FW-H3isTISRDNSKNTLYLQLNSLRAEDTAVYYC (SEQIDNO:168) FW-H4isWGQGTLVTVSS.

[0115] In some embodiments, the heavy chain variable region comprises an alternate FW-H3 sequence with an arginine to lysine mutation at R19 of SEQ ID NO:167. In some embodiments, one, two, three, or four of the framework sequences is/are an FW-H1 of SEQ ID NO:165, an FW-H12 of SEQ ID NO:166, an FW-H13 or SEQ ID NO:167 with an arginine to lysine mutation at R19, and an FW-H14 of SEQ ID NO:168.

[0116] In some embodiments, a library contains a plurality of polynucleotides, with at least one of the polynucleotides encoding an antibody light chain variable region (e.g., comprising a HVR-L1, HVR-L2, and HVR-L3). In some embodiments, the antibody light chain variable region comprises an HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264. In some embodiments, the antibody light chain variable region comprises an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the antibody light chain variable region comprises an HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274 In some embodiments, a library contains a plurality of polynucleotides that encodes at least one, at least 50, at least 100, at least 250, at least 500, at least 10.sup.3, at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least 10.sup.8, at least 10.sup.9, at least 10.sup.10, at least 10.sup.11, or at least 10.sup.12 unique sequences of antibody light chain variable regions. In some embodiments, a library contains a plurality of polynucleotides that encodes at least 10.sup.3 unique sequences of antibody light chain variable regions. In some embodiments, a library contains a plurality of polynucleotides that encodes at least 10.sup.5 unique sequences of antibody light chain variable regions. In some embodiments, a library contains a plurality of polynucleotides that encodes at least 10.sup.9 unique sequences of antibody light chain variable regions. In other embodiments, a library contains a polynucleotide that encodes one antibody light chain variable region. In some embodiments, a library contains a plurality of polynucleotides that encodes from 1 to about 10.sup.3 unique sequences of antibody light chain variable regions. In some embodiments, the antibody light chain variable region is any of the antibody light chain variable regions found in the patent application(s) filed under attorney docket number 69540-3000100, 69540-2000140, and/or 69540-2000100 (the disclosures of which are each incorporated herein by reference in their entireties). In some embodiments, the antibody light chain variable region comprises any of the HVR-L1, HVR-L2, and/or HVR-L3 sequences found in the patent application(s) filed under attorney docket number 69540-3000100, 69540-2000140, and/or 69540-2000100 (the disclosures of which are each incorporated herein by reference in their entireties).

[0117] In some embodiments, one or more of the polynucleotides of a library encode(s) full- length antibody heavy chain(s). In other embodiments, one or more of the polynucleotides of a library encode(s) heavy chain Fab fragment(s). In some embodiments, one or more of the polynucleotides of a library encode(s) single-chain variable fragment(s).

[0118] In some embodiments, a library contains a plurality of polynucleotides that encodes a plurality of unique antibodies. In some embodiments, each antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region of each antibody of the plurality comprises an identical sequence and comprises a HVR-H1, a HVR-H2 and a HVR-H3. In some embodiments, at least one or at least two of the HVR-H1 and HVR-H2 comprise an amino acid sequence selected from a HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and SEQ ID NOS:1-52 and 137-158), and a HVR-H2 sequence of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y(SEQ ID NO:202); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO:206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210); and SEQ ID NOS:53-136 and 159-164). The heavy chain HVR sequences described herein may be included in any combination in a library of the present disclosure that also includes polynucleotides encoding one or more light chain variable region(s).

[0119] In some embodiments, a library of the present disclosure includes one or more vectors encoding one or more polynucleotides (e.g., synthetic polynucleotides) of the present disclosure.

[0120] Further provided herein is a method of preparing a library, e.g., by providing and assembling the polynucleotide sequences (e.g., synthetic polynucleotide(s)) of a library of the present disclosure. Further provided herein is a method of making a library, e.g., by selecting one, two, or three heavy chain HVRs (e.g., one or two heavy chain HVRs of the present disclosure) comprising a sequence having multiple conformations and assembling polynucleotide sequences to produce a library of polynucleotides (e.g., synthetic polynucleotides) encoding a plurality of antibody heavy chain variable region sequences. In some embodiments, the antibody heavy chain variable region sequences are human antibody sequences. In some embodiments, the antibody heavy chain variable region comprises a HVR-H1, a HVR-H2 and a HVR-H3, and the HVR-H1 and/or HVR-H2 comprise an amino acid sequence selected from a HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and SEQ ID NOS:1-52 and 137-158), and a HVR-H2 sequence of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO:206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210); and SEQ ID NOS:53-136 and 159-164).

[0121] In some embodiments, at least one of the HVR-H1, HVR-H2, and HVR-H3 of the antibody heavy chain variable region adopts multiple conformations. In some embodiments, the multiple conformations can be assayed or detected using techniques known in the art, including, without limitation, structural determination (e.g., X-ray crystallography or NMR) and/or computational modeling.

[0122] Polynucleotides encoding a set of antibody light and/or heavy chain variable regions can be cloned into any suitable vector for expression of a portion or the entire light or heavy chain sequences. In some embodiments, the polynucleotide cloned into a vector allows production of a portion or the entire light or heavy chain sequence fused to all or a portion of a viral coat protein (i.e., creating a fusion protein) and displayed on the surface of a particle or cell. Several types of vectors are available and may be used to practice the present disclosure, for example, phagemid vectors. Phagemid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art. In some embodiments, the polynucleotides encoding a set of antibody light and/or heavy chain variable regions can be cloned into vectors for expression in bacterial cells for bacterial display or in yeast cells for yeast display. Exemplary vectors are described in US PG Pub. No. US20160145604. In some embodiments, the vector is a display vector comprising, from 5 to 3, a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., a surface of phage, bacteria, yeast, or mammalian cells), a restriction site, a second polynucleotide encoding a surface peptide capable of being displayed on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, a yeast outer wall protein, a bacterial outer membrane protein, a cell surface tether domain, or an adapter, or a truncation or derivative thereof. In certain embodiments, the second polynucleotide is gene Ill of filamentous phage M13, or a truncation or derivative thereof. In some embodiments, the surface peptide is for phage display, yeast display, bacterial display or mammalian display, or shuttling display there between. In some embodiments, when expressed, the amino acid sequence and the surface peptide are displayed as a fusion protein on the surface. In some embodiments, the vector further comprises a fusion tag 5 to the first restriction site or 3 to the second restriction site.

[0123] Certain aspects of the present disclosure relate to a population of cells containing vector(s) described herein. Antibody light and/or heavy chains encoded by polynucleotides generated by any of the techniques described herein, or other suitable techniques, can be expressed and screened to identify antibodies having desired structure and/or activity. Expression of the antibodies can be carried out, for example, using cell-free extracts (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cells are bacterial cells, yeast cells, or mammalian cells. Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and described in the references cited herein. Expression (e.g., from a library of the present disclosure) of polypeptides (e.g., antibody chains) in these cell types, as well as screening for antibodies of interest, are described in more detail below.

[0124] Alternatively, the polynucleotides can be expressed in an E. coli expression system, such as that described by Pluckthun and Skerra. (Meth. Enzymol., 1989, 178: 476; Biotechnology, 1991, 9: 273). The mutant proteins can be expressed for secretion in the medium and/or in the cytoplasm of the bacteria, as described by Better and Horwitz, Meth. Enzymol., 1989, 178: 476. In some embodiments, the single domains encoding V.sub.H and V.sub.L, are each attached to the 3 end of a sequence encoding a signal sequence, such as the ompA, phoA or pelB signal sequence (Lei et al., J. Bacteriol., 1987, 169: 4379). These gene fusions are assembled in a dicistronic construct, so that they can be expressed from a single vector and secreted into the periplasmic space of E. coli where they will refold and can be recovered in active form. (Skerra et al., Biotechnology, 1991, 9: 273). For example, antibody heavy chain genes can be concurrently expressed with antibody light chain genes to produce antibodies or antibody fragments.

[0125] In other embodiments, the antibody sequences are expressed on the membrane surface of a prokaryote, e.g., E. coli, using a secretion signal and lipidation moiety as described, e.g., in US20040072740; US20030100023; and US20030036092.

[0126] Alternatively, antibodies can be expressed and screened by anchored periplasmic expression (APEx 2-hybrid surface display), as described, for example, in Jeong et al., PNAS, 2007, 104: 8247 or by other anchoring methods as described, for example, in Mazor et al., Nature Biotechnology, 2007, 25: 563.

[0127] Higher eukaryotic cells, such as mammalian cells, for example myeloma cells (e.g., NS/0 cells), hybridoma cells, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells, can also be used for expression of the antibodies of the present disclosure. Typically, antibodies expressed in mammalian cells are designed to be secreted into the culture medium, or expressed on the surface of the cell. The antibody or antibody fragments can be produced, for example, as intact antibody molecules or as individual V.sub.H and V.sub.L, fragments, Fab fragments, single domains, or as single chains (scFv).

[0128] In other embodiments, antibodies can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103: 9637). In some embodiments, as described above and exemplified below, antibodies can be selected after production of a portion or the entire light or heavy chain sequence fused to all or a portion of a viral coat protein (i.e., creating a fusion protein) and displayed on the surface of a particle or cell, e.g., using phage display.

[0129] Certain aspects of the present disclosure relate to a non-human animal comprising a polynucleotide library of the present disclosure. For example, a non-human animal of the present disclosure may be modified such that its genome includes a polynucleotide encoding a heavy chain variable region of the present disclosure. In a non-limiting example, a transgenic mouse is generated that includes a heavy chain immunoglobulin locus modified to express one or more of the heavy chain variable regions of the present disclosure. In some embodiments, the transgenic animal (e.g., mouse) expresses antibodies or heavy chains encoded by the polynucleotides. Techniques for modifying one or more immunoglobulin loci of a non-human animal are known in the art (e.g., methods used to generate Xenomouse).

[0130] The screening of the antibodies derived from the libraries of the present disclosure can be carried out by any appropriate means known in the art. For example, binding activity can be evaluated by standard immunoassay and/or affinity chromatography. Screening of the antibodies of the present disclosure for catalytic function, e.g., proteolytic function can be accomplished using a standard assays, e.g., a hemoglobin plaque assay. Determining binding affinity of an antibody to a target can be assayed in vitro using a variety of well-known techniques, e.g., a BIACORE instrument, which measures binding rates of an antibody to a given target or antigen based on surface plasmon resonance, or Bio-Layer Interferometry (BLI), as exemplified below using the ForteBio Octet RED96 platform (Pall Life Sciences). In vivo assays can be conducted using any of a number of animal models and then subsequently tested, as appropriate, in humans. Cell-based biological assays are also contemplated. The antibodies or antigen binding fragments can be further selected for functional activity, for example, antagonist or agonist activity. Exemplary screening methods are described herein. For example, in some embodiments, affinity of binding between fab fragment(s) and one or more target(s) is measured using BLI by tagging antigens with human IgG1-Fc tag and capture by Anti-hIgG-Fc Capture (AHC) Biosensor. Fabs can be tagged at their C-terminus of the CH1 domain with a His6 tag, over-expressed in a host cell such as E. coli, and purified, e.g., using a Ni-NTA resin. Affinity can then be measured using AHC sensors (anti-human IgG-Fc capture dip and read biosensors) dipped into wells containing the purified fabs diluted, e.g., to 5-10 g/mL with kinetic buffer.

[0131] After binders are identified by binding to the target or antigen, and/or functional assays the nucleic acid can be extracted. Extracted DNA can then be used directly to transform E. coli host cells or alternatively, the encoding sequences can be amplified, for example using PCR with suitable primers, and sequenced by any typical sequencing method. Variable domain DNA of the binders can be restriction enzyme digested and then inserted into a vector for protein expression.

IV. Antibodies and Antibody Production

[0132] Provided herein are antibodies identified and selected from the libraries described herein. Certain aspects of the present disclosure relate to antibody light chain or heavy chain HVRs, variable regions comprising the HVRs, and/or polynucleotide(s) encoding the same. In some embodiments, the HVRs and/or variable regions are part of an antibody fragment, full- length antibody, or single-chain variable fragment (scFv).

[0133] In some embodiments, a heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula I) X1 TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO: 199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200). In some embodiments, a heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); (Formula VI) IGX11YX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207). In some embodiments, the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210). In some embodiments, a heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises the amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO: 199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula W) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); (Formula VI) IGX11YX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207). In some embodiments, a heavy chain variable region comprises an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises the amino acid sequence according to a formula selected from the group consisting of (Formula I) X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); (Formula II) YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO: 199); and (Formula III) FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising the amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210).

[0134] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207).

[0135] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210).

[0136] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207).

[0137] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210).

[0138] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and (Formula X) VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207).

[0139] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising the amino acid sequence according to the formula FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and (Formula XII) VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210).

[0140] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 and/or HVR-H2 comprise an amino acid sequence listed in Table 1 below.

TABLE-US-00002 TABLE1 HeavychainHVRsequences SEQIDNO. DesignedSequence HVR-H1 1 FTFTDYGIHWV 2 FTFTGYAIHWV 3 FTFTNYGIHWV 4 YTFSDYAIHWV 5 YTFSDYGIHWV 6 YTFSGYAIHWV 7 YTFSGYGIHWV 8 YTFSNYGIHWV 9 YTFSSYGIHWV 10 YTFSGYWIHWV 11 YTFSNYWIHWV 12 FTFSGYWIHWV 13 FTFSNYWIHWV 14 YTFSDYWIHWV 15 YSISSGHHWAWI 16 YSISSGHYWNWI 17 YSISSGHYWSWI 18 YSISSGHYWTWI 19 YSISSGYHWAWI 20 YSISSGYHWDWI 21 YSISSGYHWGWI 22 YSISSGYHWNWI 23 YSISSGYHWSWI 24 YSISSGHHWDWI 25 YSISSGYYWDWI 26 YSISSGYYWNWI 27 YSISSGYYWTWI 28 YSITSGHHWAWI 29 YSITSGHHWDWI 30 YSITSGHHWGWI 31 YSITSGHHWNWI 32 YSITSGHHWSWI 33 YSISSGHHWGWI 34 YSITSGHYWAWI 35 YSITSGHYWDWI 36 YSITSGHYWGWI 37 YSITSGHYWNWI 38 YSITSGHYWSWI 39 YSITSGYHWAWI 40 YSITSGYHWGWI 41 YSISSGHHWNWI 42 YSITSGYHWNWI 43 YSITSGYHWSWI 44 YSITSGYYWDWI 45 YSISSGHHWTWI 46 YSISSGHYWDWI 47 FSLSTSGVAVSWI 48 FSLSTGGVAVGWI 49 FSLSTGGVAVSWI 50 FSLSTGGVGVAWI 51 FSLSTGGVGVSWI 52 FSLSTSGVAVAWI 137 FTFSDYAIHWV 138 FTFSDYGIHWV 139 YTFSNYAIHWV 140 YTFSSYAIHWV 141 YTFTDYAIHWV 142 YTFTDYGIHWV 143 YTFTNYAIHWV 144 YTFTNYGIHWV 145 FTFSGYGIHWV 146 FTFSNYAIHWV 147 FTFSSYGIHWV 148 FTFSDYWIHWV 149 FTFTSYWIHWV 150 YSISSGYYWGWI 151 YSITSGYYWNWI 152 YSITSGYYWSWI 153 YSISSGHYWAWI 154 YSISSGHYWGWI 155 FSLSTSGVAVGWI 156 FSLSTSGVGVAWI 157 FSLSTSGVGVGWI 158 FSLSTGGVGVGWI HVR-H2 53 LARIDWDDDKRYSPSLKSRL 54 LALIDWDDDKRYSPSLKSRL 55 LALIDWDDDKRYSTSLKSRL 56 LALIDWDDDKYYSPSLKSRL 57 LALIDWADDKYYSPSLKSRL 58 LALIDWAGDKSYSTSLKSRL 59 LARIDWDDDKYYSPSLKSRL 60 LARIDWDDDKYYSTSLKSRL 61 LARIDWDGDKYYSTSLKSRL 62 IGDIYHSGSTYYSPSLKSRV 63 IGEIYHSGSTYYSPSLKSRV 64 IGEIYYSGSTYYSPSLKSRV 65 IGSIYHSGNTNYNPSLKSRV 66 IGEIYHSGNTYYNPSLKSRV 67 IGEIYHSGSTYYNPSLKSRV 68 IGEIYYSGSTYYNPSLKSRV 69 IGDIYHSGNTYYNPSLKSRV 70 IGDIYHSGSTYYNPSLKSRV 71 VSAISGYGDTTYYADSVKGRF 72 VSAISGYGGSTYYADSVKGRF 73 VSAISGYGGTTYYADSVKGRF 74 VSGISGAGDTTYYADSVKGRF 75 VSGISGDGDTTYYADSVKGRF 76 VSGISGDGGSTYYADSVKGRF 77 VSGISGYGDTTYYADSVKGRF 78 VSGISGYGGTTYYADSVKGRF 79 VSVISGDGDTTYYADSVKGRF 80 VSVISGYGGSTYYADSVKGRF 81 VSGISGDGSTTYYADSVKGRF 82 VSGISGYGSTTYYADSVKGRF 83 VSVISGSGSTTYYADSVKGRF 84 VSVISGYGSSTYYADSVKGRF 85 VSVISGYGSTTYYADSVKGRF 86 VSAISGYGSTTYYADSVKGRF 87 VSSISGYGDTTYYADSVKGRF 88 VSSISGYGGSTYYADSVKGRF 89 VSSISGYGGTTYYADSVKGRF 90 VSYISGAGDTTYYADSVKGRF 91 VSSISGAGDTTYYADSVKGRF 92 VSYISGAGGTTYYADSVKGRF 93 VSYISGDGDTTYYADSVKGRF 94 VSYISGDGGSTYYADSVKGRF 95 VSYISGDGGTTYYADSVKGRF 96 VSYISGSGDTTYYADSVKGRF 97 VSSISGAGGSTYYADSVKGRF 98 VSYISGYGDTTYYADSVKGRF 99 VSYISGYGGTTYYADSVKGRF 100 VSSISGAGGTTYYADSVKGRF 101 VSSISGDGDTTYYADSVKGRF 102 VSSISGDGGTTYYADSVKGRF 103 VSSISGAGSSTYYADSVKGRF 104 VSSISGAGSTTYYADSVKGRF 105 VSSISGDGSSTYYADSVKGRF 106 VSSISGDGSTTYYADSVKGRF 107 VSSISGYGSSTYYADSVKGRF 108 VSSISGYGSTTYYADSVKGRF 109 IGWINPNRGDTKYAQKFQGRV 110 IGWINPNRGDTNYAQKFQGRV 111 IGWINPNRGGTKYAQKFQGRV 112 IGWINPNRGGTNYAQKFQGRV 113 IGWINPNRGSTKYAQKFQGRV 114 IGWINPNRGSTNYAQKFQGRV 115 IGRINPNFGDTNYAQKFQGRV 116 IGWINPNFGDTNYAQKFQGRV 117 IGWINPNFGSTKYAQKFQGRV 118 IGWINPNFGSTNYAQKFQGRV 119 IGIINPNRGDTKYAQKFQGRV 120 IGIINPNRGDTNYAQKFQGRV 121 IGIINPNFGDTNYAQKFQGRV 122 IGWISPSGGGTKYAQKFQGRV 123 IGWISPSGGGTNYAQKFQGRV 124 IGWISPSSGGTKYAQKFQGRV 125 IGWISPSSGGTNYAQKFQGRV 126 IGWIYPSGGGTKYAQKFQGRV 127 IGWIYPSGGGTNYAQKFQGRV 128 IGWISPSGGSTNYAQKFQGRV 129 IGWISPSSGSTKYAQKFQGRV 130 IGWISPSSGSTNYAQKFQGRV 131 IGWISPSGGSTKYAQKFQGRV 132 IGIIYPSGGGTNYAQKFQGRV 133 IGIISPSGGGTKYAQKFQGRV 134 IGIISPSGGGTNYAQKFQGRV 135 IGIIYPSGGSTNYAQKFQGRV 136 VGRIKSKTDGYTTEYAAPVKGRF 159 VSAISGSGSTTYYADSVKGRF 160 VSSISGSGDTTYYADSVKGRF 161 VSSISGSGGSTYYADSVKGRF 162 VSSISGSGGTTYYADSVKGRF 163 VSSISGDGGSTYYADSVKGRF 164 VSSISGSGSTTYYADSVKGRF

[0141] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H3 is any HVR-H3 known in the art. In some embodiments, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0142] In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 and/or HVR-H2 are any of the HVR-H1s and/or HVR-H2s described herein. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from any HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and SEQ ID NOS:1-52 and 137-158). In some embodiments, the HVR-H2 comprises an amino acid sequence selected from any HVR-H2 of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO:206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210); and SEQ ID NOS:53-136 and 159-164).

[0143] In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52.

[0144] In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164. In some embodiments, the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136.

[0145] In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising a HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of Formula (I), Formula (II), and Formula (III), or the HVR-H2 comprises an amino acid sequence selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), Formula (X), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising a HVR-H1, a HVR-H2 and a HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 7, 8, 9, 11, 13, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 31, 33, 34, 38, 40, 42, 43, 45, 47, 49, 50, and 51, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 60, 63, 65, 66, 67, 70, 82, 89, 93, 95, 105, 109, 110, 117, 121, 122, 123, 124, 128, 129, 130, 131, 132, and 134. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 14, 15, 30, 32, 35, 37, 39, 41, 44, 46, and 48, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 56, 59, 61, 62, 64, 68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 72, 81, 83, 86, 90, 91, 99, 100, 103, 106, 107, 108, 112, 113, 116, 118, 126, 135, and 136. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, or wherein the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 57, 58, 80, 84, 85, 87, 88, 92, 94, 96, 97, 98, 101, 102, 104, 111, 114, 115, 119, 120, 125, 127 and 133.

[0146] In some embodiments, provided herein is an antibody heavy chain with a heavy chain variable region comprising an HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 comprises an amino acid sequence selected from SEQ ID NOS:1-52 and 137-158, and the HVR-H2 comprises an amino acid sequence selected from SEQ ID NOS:53-136 and 159-164. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from SEQ ID NOS:1-52, and the HVR-H2 comprises an amino acid sequence selected from SEQ ID NOS:53-136.

[0147] In certain embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VI); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VII); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (VIII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (V); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (V); and a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (VIII). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XI); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XI); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XI). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (IV); a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (X); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (IX); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (X); a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (VIII); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (X). In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of Formula (II) and a HVR-H2 comprising the amino acid sequence of Formula (XIII); a HVR-H1 comprising the amino acid sequence of Formula (I) and a HVR-H2 comprising the amino acid sequence of Formula (XIII); and a HVR-H1 comprising the amino acid sequence of Formula (III) and a HVR-H2 comprising the amino acid sequence of Formula (XIII).

[0148] The heavy chain HVR sequences described herein may be included in any combination in an antibody heavy chain or heavy chain variable region of the present disclosure. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-52 and 137-158, and a HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-52, and a HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, and an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, and an HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:223-256. In some embodiments, a heavy chain variable region comprises an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164 and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52 and 137-158, an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136 and 159-164, and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, a heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:1-52, an HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:53-136, and a HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0149] In certain embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:122; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:154, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:161; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:145, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:128; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:61; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:153, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:155, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:67; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:100; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:138, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:123; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:126; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:129; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:3 1, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:124; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:130; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:150, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:132; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:12, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:149, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:117; and a HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:134. In some embodiments, the HVR-H1 and HVR-H2 are selected from the group consisting of: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:26, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1 51, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:34, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:5, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:6, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:7, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:17, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:25, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:152, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:156, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:157, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:94; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:48, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:58; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:50, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:163; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:160; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:92; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-2 comprising the amino acid sequence of SEQ ID NO:97; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:158, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:164; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:137, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:54; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:3, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:127; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:85; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:4, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:109; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:139, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:8, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:120; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:140, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:131; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:141, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:142, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:159; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:143, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:116; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:144, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:146, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:110; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:147, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:133; a HVR-H1 comprising the amino acid sequence of SEQ ID NO:148, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; and a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13, and a HVR-H2 comprising the amino acid sequence of SEQ ID NO:118.

[0150] In some embodiments, a heavy chain variable region comprises three of a HVR-H1, a HVR-H2, and a HVR-H3, wherein the HVR-H1 and HVR-H2 are listed in Table 1. In some embodiments, the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:223-256. In some embodiments, a heavy chain variable region comprises a sequence selected from SEQ ID NOS: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence selected from SEQ ID NOS: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195.

[0151] In some embodiments, a heavy chain variable region further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4). In some embodiments, one, two, three, or four of the framework sequences is/are the following:

TABLE-US-00003 (SEQIDNO:165) FW-H1isEVQLVESGGGLVQPGGSLRLSCAASG (SEQIDNO:166) FW-H2isRQAPGKGLEW (SEQIDNO:167) FW-H3isTISRDNSKNTLYLQLNSLRAEDTAVYYC (SEQIDNO:168) FW-H4isWGQGTLVTVSS.

[0152] In some embodiments, the heavy chain variable region comprises an alternate FW-H3 sequence with an arginine to lysine mutation at R19 of SEQ ID NO:167. In some embodiments, one, two, three, or four of the framework sequences is/are an FW-H1 of SEQ ID NO:165, an FW-H12 of SEQ ID NO:166, an FW-H13 or SEQ ID NO:167 with an arginine to lysine mutation at R19, and an FW-H14 of SEQ ID NO:168.

[0153] In some embodiments, further provided herein is an antibody comprising a heavy chain and a light chain, where the heavy chain includes a heavy chain variable region of the present disclosure, and where the light chain includes any light chain variable region (e.g., comprising a HVR-L1, HVR-L2, and HVR-L3) known in the art. In some embodiments, the antibody light chain variable region comprises an HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264. In some embodiments, the antibody light chain variable region comprises an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the antibody light chain variable region comprises an HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and an HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the antibody light chain comprises any of the antibody light chain variable regions found in the patent application(s) filed under attorney docket number 69540-3000100, 69540- 2000140, and/or 69540-2000100 (the disclosures of which are each incorporated herein by reference in their entireties). In some embodiments, the antibody light chain comprises a light chain variable region comprising any of the HVR-L1, HVR-L2, and/or HVR-L3 sequences found in the patent application(s) filed under attorney docket number 69540-3000100, 69540-2000140, and/or 69540-2000100 (the disclosures of which are each incorporated herein by reference in their entireties).

[0154] IgG-derived scaffolds such as Fab and single chain Fv (scFv), as well as stabilized Fv or scFv, have been designed and prepared with the ability to specifically recognize and tightly bind antigens. Alternative protein scaffolds, or non-IgG like scaffolds, have been explored for analogous applications. Several protein families with non-Ig architecture such as the protein A, fibronectin, the ankyrin repeat, Adnectins, Affibodies, Anticalins, DARPins, engineered Kunitz inhibitors or the lipocalins, cyclic and polycyclic peptides can be empowered with novel binding sites by employing methods of combinatorial engineering, such as site-directed random mutagenesis in combination with phage display, yeast display, or other molecular selection techniques. These novel alternative binding reagents are collectively called engineered protein scaffolds, illustrating the fact that a rigid natural protein structure is used to modify an existingor to implement a newbinding site for a prescribed target using the dynamic binding motifs or units introduced here. Compared with antibodies or their recombinant fragments, these protein scaffolds often provide practical advantages including elevated stability and high production yield in microbial expression systems. As these novel binding proteins are obtained by means of a biomolecular engineering process in order to achieve tight target-binding activity, they may also be subjected to further selection schemes focused at other desired properties (such as solubility, thermal stability, protease resistance etc.). Consequently, engineered protein scaffolds have become attractive for many applications in biotechnology and biomedical research, especially for multi-specific binding motifs. The effort to generate such an alternative binding protein with beneficial properties are directed toward therapeutic use with special emphasis on biomolecular structure and function as well as on approaches toward clinical application.

[0155] In some embodiments, further provided herein is one or more polypeptides (e.g., a scaffold polypeptide, including IgG-derived scaffold polypeptides (such as Fabs, single chain Fvs, and stabilized Fvs) or non-IgG-derived scaffold polypeptides (such as protein A, fibronectin, ankyrin repeat, Adnectins, Affibodies, Anticalins, DARPins, engineered Kunitz inhibitors or the lipocalins, cyclic and polycyclic peptides)) comprising one or more HVRs described herein. In some embodiments, the polypeptide comprises an HVR-H1 comprising an amino acid sequence selected from any HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV, wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (SEQ ID NO:198); YSIX1SGX2X3WX4WI, wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T (SEQ ID NO:199); and FSLSTX1GVX2VX3WI, wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO:200); and SEQ ID NOS:1-52 and 137-158). In some embodiments, the polypeptide comprises an HVR-H2 comprising an amino acid sequence selected from any HVR-H2 of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T (SEQ ID NO:201); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:203); VSX1ISGX2GX3X4TYYADSVKGRF, wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T (SEQ ID NO:204); IGX1INPNX2GX3TX4YAQKFQGRV, wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N (SEQ ID NO:205); IGX1IX2PSX3GX4TX5YAQKFQGRV, wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO:206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO:207); IGX1IX2X3SGSTYYSPSLKSRV, wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:208); IGX1IYX2SGX3TX4YNPSLKSRV, wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y (SEQ ID NO:209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF, wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S (SEQ ID NO:210); and SEQ ID NOS:53-136 and 159-164). In some embodiments, the polypeptide comprises an HVR-H3 comprising an amino acid sequence selected from any HVR-H3 sequence of the present disclosure (e.g., SEQ ID NOs: 223-256). In some embodiments, the polypeptide comprises an HVR-L1 comprising an amino acid sequence selected from any HVR-L1 sequence of the present disclosure (e.g., SEQ ID NOs: 257-264). In some embodiments, the polypeptide comprises an HVR-L3 comprising an amino acid sequence selected from any HVR-L3 sequence of the present disclosure (e.g., SEQ ID NOs: 265-274).

[0156] In some embodiments, the polypeptide comprises two or more (e.g., two or more, three or more, four or more, or all five) of the HVR-H1 , HVR-H2, HVR-H3, HVR-L1, and/or HVR-L3 sequences described herein. In some embodiments, the polypeptide comprises two of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and/or HVR-L3 sequences described herein, wherein the two are a HVR-H1 and a HVR-H2; a HVR-H1 and a HVR-H3; a HVR-H1 and a HVR-L1; a HVR-H1 and a HVR-L3; a HVR-H2 and a HVR-H3; a HVR-H2 and a HVR-L1; a HVR-H2 and a HVR-L3; a HVR-H3 and a HVR-L1; a HVR-H3 and a HVR-L3; or a HVR-L1 and a HVR-L3. In some embodiments, the polypeptide comprises three of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and/or HVR-L3 sequences described herein, wherein the three are a HVR-H1, a HVR-H2, and a HVR-H3; a HVR-H1, a HVR-H2, and a HVR-L1; a HVR-H1, a HVR-H2, and a HVR-L3; a HVR-H1, a HVR-H3, and a HVR-L1; a HVR-H1, a HVR-H3, and a HVR-L3; a HVR-H1, a HVR-L1 and a HVR-L3; a HVR-H2, a HVR-H3, and a HVR-L1; a HVR-H2, a HVR-H3, and a HVR-L3; a HVR-H2, a HVR-L1, and a HVR-L3; or a HVR-H3, a HVR-L1, and a HVR-L3. In some embodiments, the polypeptide comprises four of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and/or HVR-L3 sequences described herein, wherein the four are a HVR-H1, a HVR-H2, a HVR-H3, and a HVR-L1; a HVR-H1, a HVR-H2, a HVR-H3, and a HVR-L3; a HVR-H1, a HVR-H2, a HVR-L1, and a HVR-L3; a HVR-H1, a HVR-H3, a HVR-L1, and a HVR-L3; or a HVR-H2, a HVR-H3, a HVR-L1, and a HVR-L3. In some embodiments, the polypeptide comprises five of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and/or HVR-L3 sequences described herein, wherein the five are a HVR-H1, a HVR-H2, a HVR-H3, a HVR-L1, and a HVR-L3.

[0157] In some embodiments, further provided herein is an antibody fragment or scFv comprising a light chain variable region and a heavy chain variable region of the present disclosure.

[0158] In some embodiments, an antibody or antibody fragment of the present disclosure binds at least 1 target (e.g., a target protein or an epitope) or at least two targets with particular binding affinities. For example, in some embodiments, an antibody or antibody fragment of the present disclosure binds at least 1 target or at least two targets with an equilibrium dissociation constant (Kd) of about 10.sup.7M or less, 10.sup.8 M or less, 10.sup.9 M or less, 10.sup.10 M or less, or 10.sup.11 M or less. In some embodiments, an antibody or antibody fragment of the present disclosure binds at least 1 target or at least two targets with an equilibrium dissociation constant (Kd) of between about 10.sup.7 and about 10.sup.11M. Exemplary assays for determining binding affinity are described and exemplified infra (See e.g., the ForteBio assay of Example 4 below).

[0159] In some embodiments, an antibody or antibody fragment of the present disclosure has a melting temperature (Tm) of at least 60 C. For example, in some embodiments, an antibody or antibody fragment of the present disclosure has a Tm of between about 60 C. and about 90 C., between about 65 C. and about 90 C., between about 70 C. and about 90 C., between about 75 C. and about 90 C., between about 80 C. and about 90 C., between about 85 C. and about 90 C., or at least about 65 C., at least about 70 C., at least about 72 C., at least about 75 C., at least about 80 C., or at least about 85 C. In some embodiments, an antibody or antibody fragment of the present disclosure has a Tm of between about 60 C. and about 90 C. Various methods of measuring Tm for an antibody or antibody fragment are known in the art. Exemplary assays for determining antibody Tm are described and exemplified infra (See e.g., the DSF assay of Example 4 below).

[0160] Antibodies of the present disclosure may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids encoding any antibody described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the V.sub.L and/or an amino acid sequence comprising the V.sub.H of the antibodies (e.g., the light and/or heavy chains of the antibodies). In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided herein. In some embodiments, a host cell comprising such nucleic acids is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the V.sub.L, of the antibody and an amino acid sequence comprising the V.sub.H of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the V.sub.L, of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the V.sub.H of the antibody. In some embodiments, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In some embodiments, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0161] For recombinant production of antibodies of the present disclosure, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0162] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and may be further purified.

[0163] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been humanized, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0164] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0165] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES technology for producing antibodies in transgenic plants).

[0166] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BEM); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO- 76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR.sup.CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0167] Bispecific Antibodies with Identical/Common/Single Heavy Chains

[0168] Further provided herein is a bispecific antibody having an identical heavy chain variable region of the present disclosure (e.g., having two light chain variable regions with different binding specificities and two identical heavy chain variable regions). In some embodiments, the bispecific antibody comprises two different light chains, wherein the first light chain comprises a kappa C.sub.L domain (e.g., a human kappa C.sub.L domain), and the second light chain comprises a lambda C.sub.L domain (e.g., a human lambda C.sub.L domain). Methods of making and/or purifying bispecific antibodies comprising a kappa C.sub.L domain and a lambda C.sub.L domain are known in the art (See e.g., Fischer et al. (2015), Nat. Commun. 6:6113; US20140179547). For example, a bispecific antibody comprising: a) two identical heavy chain variable regions (e.g., any one of the heavy chain variable regions described herein), b) a first light chain comprising a first light chain variable region and a kappa C.sub.L domain, and c) a second light chain comprising a second light chain variable region and a lambda C.sub.L domain (e.g., the constant region of a second light chain comprising a kappa C.sub.L domain is switched with a lambda C.sub.L domain) may be constructed and expressed (e.g., cloned into one or more expression vectors and expressed in one or more suitable host cells). The resulting bispecific IgG constructed in this way (e.g., comprising both a kappa and a lambda C.sub.L domain) may be purified using the following steps: first, total IgGs are recovered from the culture supernatant using protein A or IgG-CH1 Capture Select affinity chromatography, resulting in the elimination of free light chains and other contaminants; next, IgGs containing a kappa C.sub.L domain are captured using KappaSelect affinity resin, and monospecific IgGs with light chains containing only lambda C.sub.L domains are eliminated in the column flow through; finally, pure bispecific kappa-lambda-bodies are recovered using LambdaFabSelect affinity resin, and separated from the monospecific IgGs with light chains containing only kappa C.sub.L domains that do not bind to the resin. Alternatively, the bispecific common heavy chain IgG (e.g., as described above) can be purified by protein A and resolved using resins specific to each light chain C.sub.L domain based on differences in one or more biophysical properties of the differing light chains (such as different molecular weights, different isoelectric points (pI), etc.).

[0169] In some embodiments, the bispecific antibody comprises two antibody light chain variable regions and two identical heavy chain variable regions, where the bispecific antibody includes: a first binding domain that binds to a first target or antigen and comprises a first antibody light chain variable region and a first heavy chain variable region; and a second binding domain that binds to a second target or antigen and comprises a second antibody light chain variable region and a second antibody heavy chain variable region; where the second antibody heavy chain variable region has a sequence identical to the first antibody heavy chain variable region sequence. In some embodiments, the first and second binding domains bind to different target biomolecules. In some embodiment, the first and second binding domains bind to different epitopes on a same biomolecule. In some embodiments, the first antibody heavy chain variable region is part of a first antibody heavy chain comprising the first heavy chain variable region and a first heavy chain constant region (e.g., comprising CH1, hinge, CH2 and CH3). In some embodiments, the second antibody heavy chain variable region is part of a second antibody heavy chain comprising the second heavy chain variable region and a second heavy chain constant region (e.g., comprising CH1, hinge, CH2 and CH3). In some embodiments, the first antibody light chain variable region is part of a first antibody light chain comprising the first light chain variable region and a first light chain constant region. In some embodiments, the second antibody light chain variable region is part of a second antibody light chain comprising the second light chain variable region and a second light chain constant region. In some embodiments, the first and the second antibody heavy chains have sequences identical to a heavy chain of the present disclosure.

[0170] Further provided herein is a method of generating a bispecific antibody having an identical heavy chain variable region of the present disclosure (e.g., having two light chain variable regions with different binding specificities and two identical heavy chain variable regions). In some embodiments, the method includes (a) selecting a first antigen binding domain that binds to a first antigen and comprises a first antibody light chain variable region and a first heavy chain variable region of the present disclosure; (b) selecting a second antigen binding domain that binds to a second antigen and comprises a second antibody light chain variable region and a second heavy chain variable region of the present disclosure, where the second antibody heavy chain variable region has a sequence identical to the first antibody heavy chain variable region sequence; and (c) producing the bispecific antibody comprising a light chain variable region comprising the amino acid sequence of the first antibody light chain variable region, a light chain variable region comprising the amino acid sequence of the second antibody light chain variable region, a heavy chain variable region comprising the amino acid sequence of the first antibody heavy chain variable region sequence, and a heavy chain variable region comprising the amino acid sequence of the second antibody heavy chain variable region sequence. In some embodiments, the first heavy chain variable region is encoded by a polynucleotide from a library of the present disclosure.

[0171] In some embodiments, bispecific antibodies described herein may have additional specificities. For example, one of the antigen or target binding sites of the bispecific antibody may bind to more than one target specifically.

[0172] Methods for making/generating bispecific antibodies are known in the art. Production of full length bispecific antibodies can be based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

V. Kits

[0173] In another aspect, provided herein is a kit comprising a library of polynucleotides of the present disclosure. In some embodiments, the kit further comprises a package insert comprising instructions for expressing, modifying, screening, or otherwise using the library, e.g., to identify an antibody HVR or variable region of interest. In some embodiments, the kit further comprises one or more buffers, e.g., for storing, transferring, transfecting, or otherwise using one or more of the polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further comprises one or more containers for storing one or more of the polynucleotides. In some embodiments, the kit further comprises one or more vectors, e.g., for transfection of a host cell with one or more of the polynucleotides.

EXAMPLES

[0174] The present disclosure will be more fully understood by reference to the following examples. The examples should not, however, be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1

Identification of the Minimal set of Dynamic Motifs on Hypervariable Regions

[0175] To understand variability of antibody variable domains at a structural level, an algorithm was developed to map the geometric alignment for antibody variable domains, and further, to calculate the structural and sequence entropy based upon the geometric alignment. Taking such an approach combines the classical theory of antibody diversity being determined by the well-established process of V(D)J recombination coupled with conformational diversity from dynamic units (template-directed conformational selection by Linus Pauling; See e.g., James, L. and Tawfik, D. Conformational diversity and protein evolutiona 60-year-old hypothesis revisited, Trends Biochem Sci. 2003 July; 28(7):361-8) to allow sampling of an almost infinite epitope space by selection and adaptation of antibody binding sites. As an example, this algorithm was used to analyze the structural and sequence variability of 113 high-resolution crystal structures of human antibody variable heavy chain domains. Entropy was calculated and plotted for every position of the variable heavy chain domain, (FIG. IA; structural entropy in bold line, sequence entropy in dotted line). The results obtained by calculating the structural and sequence entropy based upon geometric alignment were used to locate the hyper-variable (HVR) regions, and to identify the critical positions on these variable regions. For comparison, the HVRs (as defined by the methodology described above) and CDRs (as defined by Kabat) were identified for an exemplary antibody heavy chain variable domain sequence (FIG. 1B).

[0176] Interestingly, variability as assessed by structural alignments was generally lower than the variability observed with sequence alignments. While variability was generally lower as assessed by structural alignments, there were a number of sites/regions with dramatic structural variation, suggesting these variable sites may play critical roles in antibody function. Furthermore, some of those hyper-variable regions showed high flexibility with multiple conformations. The identification of regions of highly variable residues gave a more comprehensive picture of the conservation and variability of antibody variable domains that could be exploited in new antibody designs. The identification of the dynamic motif made it possible to cover a wide range of structural diversity with a reduced number of amino acid sequences. The surprising advantage of this approach to antibody design was that a more limited number of dynamic motifs could be employed in the variable regions to cover a wide range of antibody structural diversity and provide broad flexibility in these antibodies which may allow binding to multiple antigens of interest. As such, dynamic heavy chain libraries were constructed using single human germline or germline-derived sequences for the invariant residues, while a limited number of dynamic motifs (as compared to 10.sup.6, 10.sup.10 or more) were used in the hyper-variable regions HVR H1 and HVR H2 to capture the wide range of structural variability identified in these two regions.

Example 2

Construction of the Common Heavy Chain Libraries

Construction of the Heavy Chain Libraries

[0177] To begin construction of the heavy chain libraries, 3 groups of degenerate oligos were designed for the variable region HVR-H1 based on the formulas shown in Table 2, resulting in 112 unique HVR-H1 sequences. 7 groups of degenerate oligos were designed for the variable region HVR-H2 based on the formulas shown in Table 2, resulting in 565 unique HVR-H2 sequences. The synthesized degenerate oligos were converted into double stranded DNA through the following protocol: 0.75 L of 0.2 M template oligos were mixed with 10 L 5 PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M forward primer, 1 L of 100 M reverse primer, 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/L), and 33 L of water. The PCR solutions were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for six seconds) were performed, followed by extension at 72 C. for three minutes. The VH_vr1s were amplified using the primer pair F_1999 (CGTTTGTCCTGTGCAGCTTCCGG) (SEQ ID NO:211) and R_1999 (CGAGGCCCTTACCCGGGGCCTGACG) (SEQ ID NO:212), while VH vr2s were amplified using the primer pair F_2003 (CCGGGTAAGGGCCTCGAGTGG) (SEQ ID NO:213) and R_2003 (GAGCACGTCCGTTCGAATTGTCGCGACTTATAG) (SEQ ID NO:214).

[0178] The double stranded VII_vr1s and VII_vr2s were joined together through overlapping sequences at their 5 or 3 ends. The protocol used was as follows: 20 ng of VH_vr1 and 20 ng of VII_vr2 templates were mixed with 10 L 5 PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M F_1999 primer, 1 L of 100 M R 2003 primer, 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/L), and water (up to 50 L), and the mixtures were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for 10 seconds) were performed, followed by extension at 72 C. for three minutes. These PCR fragments were then purified through gel electrophoresis (GENEray Gel Extraction kit), digested with BspEI and BstBI (Thermo Scientific), and subsequently cloned into a filter vector FTV014 digested with the same two enzymes. The ligation mixture was transformed into DH10B cells by electroporation, and the number of colonies exceeding 10 fold of calculated diversity was collected for plasmid preparation. The purified plasmids constituted library VII-vr12

TABLE-US-00004 TABLE2 formulasforHVR-H1andHVR-H2designedvariantsequences X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 AminoAcidSequence Residue Residue Residue Residue Residue Residue VariantGroup Formula Identity Identity Identity Identity Identity Identity HVR-H1_A X1TFX2X3YX4IHWV F,Y S,T D,G,N, A,G,W n/a n/a (SEQIDNO:198) S HVR-H1_B YSIX1SGX2X3WX4WI S,T H,Y H,Y A,D,G, n/a n/a (SEQIDNO:199) N,S,T HVR-H1_C FSLSTX1GVX2VX3WI G,S A,G A,G,S, n/a n/a n/a (SEQIDNO:200) T HVR-H2_A LAX1IX2WX3X4DKX5Y L,R D,Y A,D,S, D,G R,S,Y P,T (SEQIDNO:201) SX6SLKSRL Y HVR-H2_B IGX1IX2X3SGSTYYSPS A,D,E, S,Y H,Y n/a n/a n/a (SEQIDNO:202) LKSRV S,Y HVR-H2_C IGX1IYX2SGX3TX4YNP D,E,R, H,Y N,S N,Y n/a n/a (SEQIDNO:203) SLKSRV S,Y HVR-H2_D VSX1ISGX2GX3X4TYY A,G,S, A,D,S, D,G,S S,T n/a n/a (SEQIDNO:204) ADSVKGRF V,Y Y HVR-H2_E IGX1INPNX2GX3TX4YA I,R,W F,R D,G,S K,N n/a n/a (SEQIDNO:205) QKFQGRV HVR-H2_F IGX1IX2PSX3GX4TX5Y I,R,W S,Y G,S D,G,S K,N n/a (SEQIDNO:206) AQKFQGRV HVR-H2_G VGRIX1SKX2X3GX4TT K,R A,T D,Y G,Y D,E P,S (SEQIDNO:207) X5YAAX6VKGRF n/a, not applicable.

[0179] Hundreds of degenerate oligos encoding the VII_vr3 with sequence diversity approaching 10.sup.5 were designed and synthesized, and converted into double strand DNA through the following protocol: 0.75 L of 0.2 M template oligos were mixed with 10 L 5 PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M forward primer, 1 L of 100 M reverse primer, 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/L), and 33 L of water. The PCR solutions were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for six seconds) were performed, followed by extension at 72 C. for three minutes. The forward primer was S1089 (ACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTG) (SEQ ID NO:215) and the reverse primer was S1090 (GAGGAGACGGTGACTAGTGTTCCTTGACCCCA) (SEQ ID NO:216). The resulting synthesized DNAs were then purified through gel electrophoresis (GENEray Gel Extraction kit), digested with AflII and Spel (Thermo Scientific), and subsequently cloned into the filter vector FTV012 digested with the same two restriction enzymes. The ligation mixture was transformed into DH10B cells by electroporation, and the number of colonies exceeding 10 fold of calculated diversity was collected for plasmid preparation. The purified plasmids constituted library VH-vr3.

[0180] To assemble the full length VH library, the purified VH-vr3 library plasmid mixture was digested with AflII and Spel (NEB), and the vr3-encoding fragments were purified through gel electrophoresis (GENEray Gel Extraction kit), and cloned into the VH-vr12 library plasmid mixture digested with the same two restriction enzymes. The ligation products were desalted (QlAquick PCR Purification Kit (QIAGEN)) before rolling circle amplification (RCA) was performed. RCA was carried out as follows: 40 ng ligation products were mixed with 10 L 10 NEBuffer 4, 50 L of 100 M pd(N)8, and water (up to 88.5 L), heated to 95 C. for three minutes, and annealed for 65 cycles (30 second each cycle) with each cycle decreasing by 1 C. The annealed reactions were incubated overnight at 30 C. after the addition of 10 L of 10 mM dNTP mix, 1 L of 100BSA, and 0.5 L of Phi29 DNA polymerase. The RCA products were first digested with Notl, DNA fragments were purified (QlAquick PCR Purification Kit), and further digested with Xhol. The digested products were then ligated with T4 DNA ligase (Thermo Scientific). After purification through ethanol precipitation, the ligation products were transformed into DH10B cells by electroporation. The purified plasmids constituted library VH-vr123. These constructs each shared the same framework regions, namely FW-H1 (SEQ ID NO:165), FW-H12 (SEQ ID NO:166), FW-H13 (SEQ ID NO:167), and FW-H4 (SEQ ID NO:168).

[0181] The above-mentioned mixtures of plasmids for the two heavy chain libraries were digested with Pvul and Acc65I, and ligated into the phagemid vector Fad40 that was also digested with the same two restriction enzymes. The ligation mixtures were transformed into DH10B cells, the resulting libraries were purified, quantified, and stored for the assembly of the complete phagemid library.

Construction of the VL Library

[0182] To begin construction of the light chain libraries, 18 groups of degenerate oligos and 5 defined oligos were designed for the variable region VL_vr1 and VL_vr2 respectively. They were converted into double stranded DNA through the following protocol: 0.75 L of 0.2 M template oligos were mixed with 10 L 5x PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M forward primer, 1 L of 100 M reverse primer, 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/ L), and 33 L of water. The PCR solutions were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for six seconds) were performed, followed by extension at 72 C. for three minutes. The VL_vr1s were amplified using the primer pair F_2898 (TACTTATGTAGGCGATCGGGTCACCATCACCTGC) (SEQ ID NO:217) and R 2898 (CGGAGCTTTTCCTGGTTTCTGTTGATAC) (SEQ ID NO:218), while VL_vr2s were amplified using the primer pair F_2013 (GAAACCAGGAAAAGCTCCGAAG) (SEQ ID NO:219) and R_2013 (CGTCCCGGAACCGGATCCAGAGAAGCGAG) (SEQ ID NO:220).

[0183] The double stranded VL_vr1s and VL_vr2s were joined together through overlapping sequences at their 5 or 3 ends. The protocol used was as follows: 20 ng of VL_vr1 and 20 ng of VL_vr2 templates were mixed with 10 L 5 PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M F_2898 primer, 1 L of 100 M R_2013 primer, 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/L), and water (up to 50 L), and the mixtures were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for 10 seconds) were performed, followed by extension at 72 C. for three minutes. These PCR fragments were then purified through gel electrophoresis (GENEray Gel Extraction kit), digested with PvuI and BamHI (Thermo Scientific), and subsequently cloned into a filter vector FTV015 digested with the same two enzymes. The ligation mixture was transformed into DH10B cells by electroporation, and the number of colonies exceeding 10 fold of calculated diversity was collected for plasmid preparation. The purified plasmids constituted library VL-vr12.

[0184] 22 groups of degenerate oligos encoding VL_vr3 were designed, synthesized, and converted into double stranded DNA through the following protocol: 0.75 L of 0.2 M template oligos were mixed with 10 L 5 PrimeSTAR buffer, 4 L dNTP mixture, 1 L of 100 M forward primer F2929 (ACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAAC) (SEQ ID NO:221), 1 L of 100 M reverse primer R2929 (GATCTCCACCTTGGTACCCTGTCCGAA) (SEQ ID NO:222), 0.5 L of PrimeSTAR HS DNA Polymerase (2.5 U/L), and 33 L of water. The PCR solutions were preheated at 96 C. for 5 minutes, then 14 cycles (96 C. for 15 seconds, 60 C. for 15 seconds, 72 C. for six seconds) were performed, followed by extension at 72 C. for three minutes. The double stranded DNAs encoding the VL_vr3 were then purified through gel electrophoresis (GENEray Gel Extraction kit), digested with Pstl and Acc65I (Thermo Scientific), and subsequently cloned into the filter vector FTV013 digested with the same two restriction enzymes. The ligation mixture was transformed into DH10B cells by electroporation, and the number of colonies exceeding 10 fold of calculated diversity was collected for plasmid preparation. The purified plasmids constituted library VL-vr3.

[0185] To assemble the full length VL library, the purified VL-vr3 library plasmid mixture was digested with PstI and Acc65I (NEB), and the vr3-encoding fragments were purified through gel electrophoresis (GENEray Gel Extraction kit), and subsequently cloned into VL-vr12 library plasmid mixture that had been digested with the same two restriction enzymes. The ligation products were transformed into DH10B cells by electroporation, and the number of colonies exceeding 10 fold of calculated diversity was collected for plasmid preparation. The purified plasmids constituted library VL-vr123. The vr123 inserts from the library plasmids VL-vr123 were then moved into the phagemid vector Fad40, using the restriction enzymes Pvul and Acc65I. The size of the library containing Fad40-vr123 reached 4*10.sup.7.

Construction of the Complete Dynamic Library

[0186] The dynamic library was composed of the heavy chain library derived from the VH-vr123 library and the light chain library derived from the Fad40-vr123 library. Both the VII-vr123 library plasmids and the Fad40-vr123 library plasmids were digested with BspEI and SpeI (Thermo Scientific). The DNA fragments encoding the heavy chain derived from the VH-vr123 library were cloned into the vector backbones derived from Fad40-vr123 library. The ligation products were desalted (QlAquick PCR Purification Kit (QIAGEN)) before rolling circle amplification (RCA). RCA was carried out as follows: 40 ng ligation products were mixed with 10 L 10 NEBuffer 4, 50 L of 100 M pd(N)8, and water (up to 88.5 L), heated to 95 C. for three minutes, and annealed for 65 cycles (30 second each cycle) with each cycle decreasing by 1 C. The annealed reactions were incubated overnight at 30 C. after the addition of 10 L of 10 mM dNTP mix, 1 L of 100BSA, and 0.5 L of Phi29 DNA polymerase. The RCA products were first digested with Notl, DNA fragments were purified (QIAquick PCR Purification Kit), and further digested with Acc65I. The digested products were then ligated with T4 DNA ligase (Thermo Scientific). After purification through ethanol precipitation, the ligation products were transformed into ER2738 cells by electroporation. A total number of 1.4*10.sup.10 colonies were collected from plates (2xYT, 1% glucose, 100 g/mL ampicillin) to make the DPL6 library.

Example 3

Screening the Common Heavy Chain Libraries to Isolate Antibodies of Interest

Preparation of Dynamic Library Phagemid Particles

[0187] To prepare the dynamic library phagemid particles for antigen panning, 5.0 liters of ER2738 cells harboring the dynamic library (described in Example 2 above) were inoculated in media containing 2xYT, 2% glucose, 100 g/mL ampicillin and 12.5 g/mL tetracycline at a starting OD.sub.600 of 0.1. The cultures were grown at 37 C., shaking at 250 rpm, until they reached OD.sub.600 of 0.6-0.8. The cells were then infected with M13K07 helper phages at a multiplicity of infection (MOI) of 10 for 30 minutes at 37 C. The infected ER2738 cells were grown overnight at 22 C. in 3.2 liters of media containing 2xYT, 100 g/mL ampicillin and 50 g/mL kanamycin. Culture supernatants were then harvested by centrifugation at 10,000 rpm for 15 minutes, and filtered through a 0.45 m low-binding membrane filter (Corning). The phagemid particles were then precipitated from the filtered supernatant using PEG/NaCl, and resuspended in PBS. An additional round of PEG/NaCl precipitation, followed by resuspension in PBS, was conducted. Phage titers were determined by OD.sub.268 measurement (assuming 1 unit at OD.sub.268 is approximately 1*10.sup.13 phage particles/mL) and confirmed by plaque assay. Library phagemid particles were stocked in 20% glycerol at 80 C.

Phage Library Panning

[0188] Antigen proteins at a concentration of 1-30 g/ml were coated on Maxisorp strips (Thermo Scientific, Cat. No. 446469) overnight at 4 C. Multiple wells of antigens were prepared for each library. The coated wells were first blocked with 5% milk in PBS for 1-2 hours at room temperature and washed with PBS. Then 1,100 L/well of phagemid particle solution (typically 1-5*10.sup.12 phages in 2% milk-PBS) was added into 4 parallel wells and incubated for 1-2 hours. Wells were then washed several times with PBS with increasing concentrations of Tween 20 (from 0.1% to 0.3%), and finally with PBS alone. The bound phagemid particles were eluted from the wells with 100 L of 0.2 M glycine-HCl for 10 minutes at room temperature. The eluted phages were immediately neutralized with 18 L of 1M Tris-HCl (pH 9.1)

[0189] Alternatively, phagemid library panning was performed using Dynabeads (M280, Streptavidin, Invitrogen, Cat. No. 60210) through KingFisher (Thermo Scientific) according to the manufacturer's instructions. 300 L of Dynabeads were washed with PBS and incubated with biotinylated anti-human Fc for 20 minutes at room temperature. The beads were then blocked with 5% BSA in PBS for one hour at room temperature. Fc-fusion antigens (70-100 pmols) were captured by one hour incubation at room temperature. The beads were then washed once with PBS, and incubated with 1 mL of phage library solution (typically 5*10.sup.12 to 1*10.sup.13 phage particles in 5% BSA-PBS) for 1-2 hours. The beads were then washed several times with PBS/Tween (0.1% to 0.3%) and PBS, and the bound phages were eluted from the beads with 100 L of 0.2 M glycine-HC1 for 10 minutes at room temperature. The eluted phages were immediately neutralized with 18 L of 1 M Tris-HCl (pH 9.1). A total of three or four rounds of panning were conducted against each of the antigens, and more than 10 fold excess of purified human Fc was included to reduce background binding.

[0190] For some of the antigens tested, 2 mL of antigens (10-30 g/mL) were used to coat immune-tubes overnight at 4 C. The volume of blocking, washing, and elution solutions were increased accordingly.

Amplification of Enriched Phage

[0191] The eluted, enriched phage pool was further amplified as follows: ER2738 cells were infected with the eluted phagemid particles at 37 C. for 30 minutes. The infected cells were then plated out on 2xYT agar plates with 2% glucose, 100 g/mL ampicillin and 12.5 g/mL tetracycline. The colonies were harvested from plates, grown in 100 ml of 2% glucose, 100 g/mL ampicillin and 12.5 g/mL tetracycline, and infected with M13K07 helper phage. The amplified phages were purified and quantified by the processes described above. Usually, the eluted phages after the final round of panning were used to infect ER2738 cells, and the resulting ER2738 colonies were picked for supernatant ELISA screening assays.

Supernatant Sandwich Elisa Assay

[0192] A sensitive sandwich Elisa assay was developed to measure the Fabs present in bacterial supernatant. Microplates were coated with polyclonal anti-human IgG (Fab specific) (Sigma 15260) to capture Fabs present in the bacterial supernatant, and then HRP labeled goat anti-human Fc was used to detect the amount of Fabs captured. The A.sub.450 of each well was measured to determine the Fab binding activity. The primary hits were defined as those whose ELISA signals were at least twice that of background, and were further characterized in the following example (Example 4).

[0193] Twelve human targets (TAGT-1, TAGT-2, TAGT-3, TAGT-4, TAGT-5, TAGT-6, TAGT-7, TAGT-8H, TAGT-9, TAGT-10H, TAGT-11, and TAGT-12), as well as two corresponding mouse targets (TAGT-8M and TAGT-10M), were screened with the constructed libraries. With these 14 antigens, a total of 690 unique positive hits with high affinity were identified. Most of the variant groups (Table 2) could form antibodies that bound to different target antigens, or were cross reactive between two species (e.g., bound TAGT-8H and TAGT-8M). The variant groups from confirmed binders were subsets of the designed variant groups shown in Table 2. A majority of the designed variants were also found in the confirmed binders (Table 3). (See the designed formulas of Table 2 vs. the formulas from the positive hits of Table 3).

TABLE-US-00005 TABLE3 formulasforHVR-H1andHVR-H2designedvariantsequencesfrompositivehits X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 AminoAcidSequence Residue Residue Residue Residue Residue Residue VariantGroup Formula Identity Identity Identity Identity Identity Identity HVR-H1_1 X1TFX2X3YX4IHWV F,Y S,T D,G,N, A,G,W n/a n/a (SEQIDNO:198) S HVR-H1_2 YSIX1SGX2X3WX4WI S,T H,Y H,Y A,D,G, n/a n/a (SEQIDNO:199) N,S,T HVR-H1_3 FSLSTX1GVX2VX3WI G,S A,G A,G,S, n/a n/a n/a (SEQIDNO:200) T HVR-H2_1 LAX1IX2WX3X4DKX5Y L,R D,Y A,D,S, D,G R,S,Y P,T (SEQIDNO:201) SX6SLKSRL Y HVR-H2_2 IGX1IX2X3SGSTYYSPS A,D,E S,Y H,Y n/a n/a n/a (SEQIDNO:208) LKSRV HVR-H2_3 IGX1IYX2SGX3TX4YNP D,E,S H,Y N,S N,Y n/a n/a (SEQIDNO:209) SLKSRV HVR-H2_4 VSX1ISGX2GX3X4TYY A,G,S, A,D,S, D,G,S S,T n/a n/a (SEQIDNO:204) ADSVKGRF V,Y Y HVR-H2_5 IGX1INPNX2GX3TX4YA I,R,W F,R D,G,S K,N n/a n/a (SEQIDNO:205) QKFQGRV HVR-H2_6 IGX1IX2PSX3GX4TX5Y I,R,W S,Y G,S D,G,S K,N n/a (SEQIDNO:206) AQKFQGRV HVR-H2_7 VGRIX1SKX2X3GX4TT K,R A,T D,Y G,Y P,S n/a (SEQIDNO:210) EYAAX5VKGRF n/a, not applicable.

Example 4

Characterization of Antibodies In Vitro

[0194] The Fabs corresponding to the primary hits identified in Example 3 above, which were tagged at their C-terminus of the CH1 domain with a His6 tag, were over-expressed in E. coli, and were purified through Ni-NTA resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Their affinities were measured by the ForteBio Octet RED96 System. Briefly, the AHC sensors (anti-human IgG-Fc capture dip and read biosensors) were used to capture antigen Fc-His fusion protein (Sino Biological #10039-H03H) were used, and dipped into wells containing the purified Fabs that were diluted to 5-10 g/mL with kinetic buffer (See also, ForteBio, Anti-human IgG Capture (AHC) Biosensors, Product Insert 41-0072-PD (2008); Yang et al. (2016), Anal. Biochem. 508:78-96). The acquired ForteBio data were processed with Data Acquisition software 7.1, and kinetic data were fitted to a 1:1 Langmuir binding model. Fab melting temperatures were measured by Differential Scanning Fluorimetry (DSF) assay. Briefly, the temperature and fluorescence monitoring was done using a qPCR machine (real time PCR). SYPRO Orange was diluted from a 5000 stock 50 fold to 100 with PBS buffers; 16 l of each Fab (0.5 mg/ml) was added to each well in a 96-well microplate and mixed with 4 l of 100 SYPRO Orange. A LightCycler480 System was used to measure fluorescence intensity. The excitation wavelength was set at 483 nm, and the emission wavelength was set at 568 nm. The temperature was increased from 25 C. to 90 C. at an increment of 1.2 to 1.3 C. per minute, and an equilibration time of 15 seconds at each measurement temperature was applied. The data were analyzed using the LightCycler480 Software. The midpoint of hydrophobic exposure, Tm, was defined as the temperature corresponding to the maximum value of the first derivative of the first fluorescence transition. (See also, Lavinder et al. (2009), J. Am. Chem. Soc.131: 3794-3795; Ericsson et al. (2006), Analytical Biochemistry 357: 289-298; Phillips and Hernandez de la Pena (2011), Current Protocols in Mol. Biol. 94: 10.28.1-10.28.15).

[0195] The 12 human target antigens (TAGT-1, TAGT-2, TAGT-3, TAGT-4, TAGT-5, TAGT-6, TAGT-7, TAGT-8H, TAGT-9, TAGT-10H, TAGT-11 and TAGT-12) were unrelated proteins sharing sequence identity lower than 26%. The sequence identity between human antigen TAGT-8H and mouse antigen TAGT-8M was 70%, while the sequence identity between human antigen TAGT-1011 and mouse antigen TAGT-10M was 60%. Multiple antibodies targeting 14 different antigens with high affinity could be successfully identified and selected from the dynamic libraries. The affinities of most binders were in the nanomolar range, and some even reached the sub-nanomolar range (FIG. 2A). In addition, the confirmed binders demonstrated good stability, with Tm ranges shown in FIG. 2B.

Example 5

Application of the Dynamic Heavy Chain Libraries

[0196] To further examine the robustness and flexibility of the heavy chain libraries, the libraries were screened against the 14 target antigens described in Example 4 above by pairing the heavy chains with different light chain libraries having a diversity varying from 10.sup.7 to 280, and all the way to a single light chain (i.e., a common light chain). The limit of the diversity design in both the heavy and light chain libraries was explored by trimming the physical size of their respective pairing partners (e.g., light chain libraries with a diversity ranging from 10.sup.7 to 280, 20, and to a single light chain) while exploring the flexibility and/or the dynamic diversity of the light chain itself. The capacity of these dynamic light chain libraries in pairing with the dynamic heavy chain libraries provided a strong rational for the library design when generating and engineering the diverse antibody hits/leads against known and challenging target antigens. Positive hits having high affinity were identified from each of the libraries tested, and a total of 690 unique positive hits were measured and confirmed with affinity data (Table 4). Their ability for binding different targets, as well as their epitope variation (including, but not limited to, the fine differences in epitope recognition between two species, as shown by the cross-species reactivity with human and murine targets with sequence identity around 60%) were examined. Positive hits using each combination of HVR-H1_1, HVR-H1_2, or HVRH-1_3, and HVR-H2_1, HVR-H2_2, or HVRH-2_3, HVR-H2_4, HVR-H_5, HVRH-2_6, or HVR-H2_7 were observed. These results indicate the power and potential of using these dynamic hypervariable region units for making antibody and protein libraries that recognize a wide range of targets for therapeutic, diagnostic and/or research reagents when they are grafted on or designed into antibody (and/or alternative protein) scaffolds. The dynamic nature of these heavy chain hypervariable region units in designing and constructing antibody (and/or non-antibody) scaffolds, when paired with light chain libraries having a wide diversity (e.g., ranging from 10.sup.7 to 280, down to a single unique sequence), is a strong validation of the dynamic antibody design concept for creating novel binding reagents useful in therapeutic, diagnostic and/or research settings.

TABLE-US-00006 TABLE 4 Affinity data for confirmed hits HVR-H1 and H2 Usage Hit ID Target ID Kd (M) HVR-H1_1 and 3757 TAGT-6 1.84E08 HVR-H2_6 3762 TAGT-6 3.04E08 3780 TAGT-8 1.47E09 3865 TAGT-11 9.48E09 3869 TAGT-11 2.35E08 3898 TAGT-11 1.83E08 4030 TAGT-8 4.90E09 4033 TAGT-8 8.75E10 4043 TAGT-8 2.69E09 4050 TAGT-10 1.65E08 4084 TAGT-8 2.94E09 4101 TAGT-8 2.12E09 4103 TAGT-8 3.59E10 4163 TAGT-8 1.37E08 4614 TAGT-8 3.53E10 4615 TAGT-8 2.28E10 4617 TAGT-8 2.88E10 4618 TAGT-8 1.08E09 4620 TAGT-8 3.48E10 4622 TAGT-8 2.74E10 4623 TAGT-8 4.85E10 4624 TAGT-8 1.00E12 4625 TAGT-8 4.02E10 4627 TAGT-8 1.82E10 4630 TAGT-8 2.67E10 4631 TAGT-8 1.83E10 4633 TAGT-8 3.22E10 4634 TAGT-8 2.07E10 4638 TAGT-8 3.14E10 4642 TAGT-8 1.89E10 4644 TAGT-8 2.48E10 4645 TAGT-8 2.96E10 4650 TAGT-8 3.57E10 4651 TAGT-8 3.01E10 4652 TAGT-8 2.94E10 4653 TAGT-8 3.27E10 4654 TAGT-8 2.32E10 4658 TAGT-8 1.42E10 4659 TAGT-8 2.12E10 4661 TAGT-8 1.62E09 4662 TAGT-8 8.98E10 4665 TAGT-8 3.69E10 4666 TAGT-8 1.17E09 4668 TAGT-8 5.79E10 4670 TAGT-8 8.21E10 4673 TAGT-8 3.23E10 4674 TAGT-8 5.02E10 4675 TAGT-8 1.00E12 4676 TAGT-8 1.62E10 4678 TAGT-8 5.98E10 4681 TAGT-8 5.43E10 4683 TAGT-8 8.97E10 4684 TAGT-8 6.69E10 4685 TAGT-8 4.78E10 4686 TAGT-8 4.78E10 4687 TAGT-8 4.08E10 4689 TAGT-8 1.63E10 4690 TAGT-8 4.67E10 4792 TAGT-10 7.39E09 5103 TAGT-10 2.67E09 5149 TAGT-11 2.91E09 5159 TAGT-11 4.09E09 5160 TAGT-11 8.07E09 5162 TAGT-11 9.87E09 5163 TAGT-11 1.71E08 5165 TAGT-11 4.06E09 5709 TAGT-11 1.93E08 5740 TAGT-11 7.26E09 5752 TAGT-11 6.33E09 5935 TAGT-12 8.78E09 5970 TAGT-12 1.35E08 5994 TAGT-12 1.58E08 5997 TAGT-12 8.51E09 6008 TAGT-12 5.10E08 6032 TAGT-2 1.63E08 6531 TAGT-3 1.08E08 7030 TAGT-8 3.47E08 7035 TAGT-8 3.04E09 7038 TAGT-8 2.33E08 7043 TAGT-8 1.34E08 7044 TAGT-8 1.12E09 7045 TAGT-8 1.11E09 7055 TAGT-8 7.57E10 7213 TAGT-12 8.87E09 7215 TAGT-12 1.61E08 7222 TAGT-12 1.26E09 7231 TAGT-12 3.38E09 7232 TAGT-12 8.06E09 7243 TAGT-12 4.95E09 7357 TAGT-3 6.14E08 BH3002 TAGT-8 2.51E10 BH3004 TAGT-8 3.00E10 BH3005 TAGT-8 3.46E10 BH3006 TAGT-8 1.94E10 HVR-H1_1 and 4025 TAGT-8 2.89E09 HVR-H2_5 4031 TAGT-8 1.06E09 4054 TAGT-10 1.58E08 4055 TAGT-10 1.07E08 4060 TAGT-10 1.10E08 4061 TAGT-10 3.42E08 4065 TAGT-10 4.31E08 4066 TAGT-10 4.76E08 4181 TAGT-10 4.27E08 4182 TAGT-10 4.24E09 4693 TAGT-10 4.87E10 4696 TAGT-10 4.58E10 4697 TAGT-10 6.21E10 4698 TAGT-10 5.70E10 4700 TAGT-10 2.62E10 4701 TAGT-10 5.60E10 4702 TAGT-10 5.02E10 4703 TAGT-10 2.85E10 4704 TAGT-10 6.65E10 4705 TAGT-10 3.02E10 4706 TAGT-10 2.50E10 4707 TAGT-10 4.29E10 4708 TAGT-10 5.29E10 4710 TAGT-10 6.26E10 4714 TAGT-10 4.46E10 4717 TAGT-10 4.61E10 4718 TAGT-10 5.32E10 4722 TAGT-10 7.46E10 4725 TAGT-10 4.84E10 4729 TAGT-10 8.80E10 4731 TAGT-10 4.67E10 4732 TAGT-10 3.33E10 4738 TAGT-10 5.34E10 4741 TAGT-10 1.66E09 4743 TAGT-10 7.40E09 4744 TAGT-10 3.73E10 4748 TAGT-10 3.92E10 4749 TAGT-10 2.55E10 4750 TAGT-10 7.86E10 4752 TAGT-10 3.34E09 4753 TAGT-10 3.43E10 4759 TAGT-10 6.59E10 4766 TAGT-10 4.09E10 4788 TAGT-10 2.88E10 4794 TAGT-10 5.56E10 4798 TAGT-10 4.35E09 4803 TAGT-10 1.88E10 4805 TAGT-10 4.26E10 4808 TAGT-10 8.28E10 4909 TAGT-10 2.90E10 5126 TAGT-8 9.54E09 5129 TAGT-8 1.12E09 5132 TAGT-8 3.06E09 5145 TAGT-8 7.00E09 5295 TAGT-9 2.21E09 6179 TAGT-10 1.99E09 6180 TAGT-10 6.11E09 6183 TAGT-10 2.70E09 6184 TAGT-10 <1.0E12 6185 TAGT-10 1.57E09 6187 TAGT-10 2.74E08 6188 TAGT-10 8.76E09 6189 TAGT-10 2.38E10 6190 TAGT-10 2.55E09 6191 TAGT-10 6.58E11 6193 TAGT-10 3.18E09 6194 TAGT-10 2.49E10 6195 TAGT-10 4.30E09 6196 TAGT-10 <1.0E12 6197 TAGT-10 8.56E09 6198 TAGT-10 2.85E09 6202 TAGT-10 1.03E09 6203 TAGT-10 1.05E08 6204 TAGT-10 6.46E09 6206 TAGT-10 3.44E09 6208 TAGT-10 3.50E09 6209 TAGT-10 3.35E09 6210 TAGT-10 5.17E10 6212 TAGT-10 2.25E09 6214 TAGT-10 1.51E09 6216 TAGT-10 6.58E10 6217 TAGT-10 4.99E09 6219 TAGT-10 3.15E09 6220 TAGT-10 3.45E09 6539 TAGT-4 3.45E09 7025 TAGT-8 4.87E08 7036 TAGT-8 1.59E08 7037 TAGT-8 2.10E08 7047 TAGT-8 2.15E08 7066 TAGT-8 1.80E08 7067 TAGT-8 3.41E08 7068 TAGT-8 1.11E08 7073 TAGT-8 3.19E09 HVR-H1_3 and 4074 TAGT-6 1.95E08 HVR-H2_4 4131 TAGT-6 <1.0E12 4132 TAGT-6 <1.0E12 4200 TAGT-6 5.68E08 4216 TAGT-6 2.59E08 4878 TAGT-12 4.07E09 5291 TAGT-1 6.57E09 5312 TAGT-6 4.50E07 5326 TAGT-6 7.84E07 5345 TAGT-6 1.02E08 5346 TAGT-6 1.61E08 5347 TAGT-6 1.21E08 5348 TAGT-6 1.02E08 5355 TAGT-6 8.71E10 5364 TAGT-6 7.26E09 5367 TAGT-6 1.49E08 5371 TAGT-6 3.97E09 5405 TAGT-6 1.01E08 5415 TAGT-6 1.64E08 5417 TAGT-6 4.04E08 5418 TAGT-6 2.02E08 5905 TAGT-12 3.83E08 5910 TAGT-12 3.30E08 5911 TAGT-12 3.35E08 5912 TAGT-12 1.68E08 5914 TAGT-12 3.30E08 5915 TAGT-12 1.82E08 5918 TAGT-12 3.46E08 5919 TAGT-12 2.38E08 5920 TAGT-12 1.88E08 5922 TAGT-12 1.95E08 5923 TAGT-12 1.60E08 5927 TAGT-12 4.35E08 5929 TAGT-12 3.20E08 5961 TAGT-12 2.41E08 5962 TAGT-12 8.06E08 5963 TAGT-12 2.07E08 5964 TAGT-12 1.40E08 5974 TAGT-12 5.02E08 5976 TAGT-12 2.88E08 5977 TAGT-12 2.70E08 5978 TAGT-12 3.25E08 5996 TAGT-12 2.21E08 5999 TAGT-12 6.29E08 6000 TAGT-12 7.86E08 6004 TAGT-12 5.50E08 6543 TAGT-3 6.78E08 7077 TAGT-6 1.88E08 7078 TAGT-6 2.52E08 7079 TAGT-6 2.99E08 7080 TAGT-6 2.44E08 7081 TAGT-6 4.31E08 7087 TAGT-6 6.96E08 7088 TAGT-6 4.36E08 7090 TAGT-6 5.55E08 7100 TAGT-6 3.50E08 7105 TAGT-6 3.33E08 7107 TAGT-6 1.22E07 7109 TAGT-6 3.20E08 7120 TAGT-6 3.45E08 7128 TAGT-6 3.97E08 7131 TAGT-6 3.04E08 7133 TAGT-6 4.03E08 7135 TAGT-6 3.17E08 7190 TAGT-6 1.03E08 7201 TAGT-6 3.26E08 7209 TAGT-12 9.36E09 7210 TAGT-12 9.85E09 7211 TAGT-12 1.26E08 7216 TAGT-12 1.88E08 7218 TAGT-12 1.49E08 7219 TAGT-12 1.44E08 7220 TAGT-12 9.12E09 7225 TAGT-12 9.53E09 7226 TAGT-12 7.57E09 7235 TAGT-12 2.18E08 7237 TAGT-12 2.13E08 7240 TAGT-12 1.17E08 7241 TAGT-12 6.43E09 7242 TAGT-12 1.71E08 7245 TAGT-12 1.38E08 7246 TAGT-12 6.22E09 7247 TAGT-12 8.93E09 7251 TAGT-12 2.69E08 7252 TAGT-12 9.56E09 7253 TAGT-12 1.62E08 7255 TAGT-12 1.20E08 7256 TAGT-12 7.08E09 7257 TAGT-12 1.11E08 7420 TAGT-9 1.38E08 7425 TAGT-9 1.77E08 HVR-H1_2 and 3761 TAGT-6 9.65E08 HVR-H2_4 3763 TAGT-6 9.30E09 4029 TAGT-8 1.89E09 4034 TAGT-8 4.27E09 4045 TAGT-8 1.10E09 4073 TAGT-6 <1.0E12 4075 TAGT-6 <1.0E12 4076 TAGT-6 7.44E09 4077 TAGT-6 <1.0E12 4123 TAGT-6 5.98E09 4124 TAGT-6 4.43E09 4125 TAGT-6 <1.0E12 4126 TAGT-6 7.27E09 4127 TAGT-6 <1.0E12 4129 TAGT-6 <1.0E12 4133 TAGT-6 3.90E10 4135 TAGT-6 <1.0E12 4137 TAGT-6 <1.0E12 4140 TAGT-6 <1.0E12 4141 TAGT-6 <1.0E12 4201 TAGT-6 1.41E08 4217 TAGT-6 9.67E08 4218 TAGT-6 2.85E08 4222 TAGT-6 5.55E08 4816 TAGT-12 5.32E09 4842 TAGT-12 4.01E10 4895 TAGT-7 6.20E09 4903 TAGT-12 1.91E09 5212 TAGT-1 9.19E09 5218 TAGT-1 6.04E09 5225 TAGT-1 3.10E10 5235 TAGT-1 1.41E08 5236 TAGT-1 1.49E08 5272 TAGT-1 2.49E08 5275 TAGT-1 9.65E09 5282 TAGT-1 1.07E08 5298 TAGT-6 3.41E07 5301 TAGT-6 2.61E07 5316 TAGT-6 1.14E08 5317 TAGT-6 3.34E07 5320 TAGT-6 6.13E07 5321 TAGT-6 7.16E07 5328 TAGT-6 3.42E07 5329 TAGT-6 2.84E06 5336 TAGT-6 6.04E07 5341 TAGT-6 2.93E08 5349 TAGT-6 6.20E09 5351 TAGT-6 7.29E09 5357 TAGT-6 7.14E09 5360 TAGT-6 2.41E08 5363 TAGT-6 9.87E09 5369 TAGT-6 2.05E08 5399 TAGT-9 3.62E08 5403 TAGT-6 8.26E09 5408 TAGT-6 2.36E08 5409 TAGT-6 1.70E08 5411 TAGT-6 1.25E08 5416 TAGT-6 1.09E08 5420 TAGT-6 1.41E08 5431 TAGT-9 1.19E08 5437 TAGT-9 1.92E08 5694 TAGT-11 9.45E09 5716 TAGT-11 8.14E09 5732 TAGT-11 5.24E09 5906 TAGT-12 1.50E08 5926 TAGT-12 3.23E08 5933 TAGT-12 3.13E08 5983 TAGT-12 2.09E08 5992 TAGT-12 1.70E08 5993 TAGT-12 1.13E08 5995 TAGT-12 1.42E08 6473 TAGT-4 2.30E08 6555 TAGT-3 4.18E08 7097 TAGT-6 2.43E08 7183 TAGT-6 1.48E08 7262 TAGT-5 2.63E09 7264 TAGT-5 3.17E09 7312 TAGT-5 3.11E09 7315 TAGT-5 5.15E09 7426 TAGT-9 1.12E08 7427 TAGT-9 5.58E09 HVR-H1_1 and 3760 TAGT-6 1.26E08 HVR-H2_4 4048 TAGT-10 3.24E09 4049 TAGT-10 9.37E09 4051 TAGT-10 1.80E08 4056 TAGT-10 1.09E08 4058 TAGT-10 1.13E08 4062 TAGT-10 2.11E08 4063 TAGT-10 1.90E08 4067 TAGT-10 1.97E08 4080 TAGT-6 <1.0E12 4130 TAGT-6 1.00E09 4138 TAGT-6 1.60E08 4139 TAGT-6 1.65E09 4723 TAGT-10 9.11E10 4733 TAGT-10 3.05E10 4734 TAGT-10 5.72E10 4767 TAGT-10 2.77E10 4771 TAGT-10 7.23E10 4797 TAGT-10 5.63E10 4807 TAGT-10 1.17E09 4829 TAGT-12 3.36E09 5194 TAGT-1 1.29E08 5200 TAGT-1 1.53E08 5210 TAGT-1 3.41E09 5297 TAGT-6 1.77E06 5300 TAGT-6 1.53E08 5315 TAGT-6 2.10E06 5353 TAGT-6 1.61E08 5354 TAGT-6 4.96E09 5438 TAGT-9 9.30E09 5510 TAGT-2 2.62E09 5513 TAGT-2 1.07E09 5526 TAGT-2 1.54E09 5528 TAGT-2 4.55E09 5532 TAGT-2 3.65E09 5553 TAGT-2 6.83E09 5554 TAGT-2 2.88E09 5557 TAGT-2 3.24E09 5558 TAGT-2 2.43E09 5561 TAGT-2 1.64E08 5565 TAGT-2 3.02E09 5568 TAGT-2 1.14E09 5600 TAGT-2 5.33E09 5612 TAGT-2 7.85E09 5614 TAGT-2 5.29E09 5622 TAGT-2 3.06E09 5642 TAGT-2 3.84E09 5710 TAGT-11 1.01E08 5739 TAGT-11 1.29E08 5745 TAGT-11 1.06E08 5746 TAGT-11 5.00E09 5754 TAGT-11 9.52E09 6221 TAGT-10 6.92E10 6471 TAGT-4 3.05E08 6536 TAGT-4 2.03E09 6537 TAGT-4 1.85E09 6540 TAGT-4 8.08E09 7204 TAGT-5 2.33E09 7212 TAGT-12 1.70E08 7260 TAGT-5 2.30E09 7271 TAGT-5 3.13E08 7276 TAGT-5 1.02E08 7311 TAGT-5 9.20E09 7317 TAGT-5 2.02E08 7323 TAGT-5 3.23E09 7365 TAGT-5 1.82E09 7366 TAGT-5 3.76E09 7369 TAGT-5 2.46E09 7371 TAGT-5 2.31E08 7373 TAGT-5 5.13E09 7374 TAGT-5 1.97E08 7378 TAGT-5 5.66E09 7411 TAGT-4 3.82E08 7415 TAGT-4 9.33E08 7418 TAGT-9 3.41E08 7419 TAGT-9 1.72E08 7429 TAGT-9 2.12E08 7431 TAGT-9 3.53E08 HVR-H1_2 and 4027 TAGT-8 1.55E09 HVR-H2_6 4027 TAGT-8M 3.81E09 4032 TAGT-8 5.11E09 4032 TAGT-8M 4.84E09 4038 TAGT-8 2.98E09 4204 TAGT-10 6.83E09 4204 TAGT-10M 6.89E09 4813 TAGT-12 2.45E10 4828 TAGT-12 1.10E09 4849 TAGT-12 8.40E10 4850 TAGT-12 1.23E09 4874 TAGT-12 4.19E09 4925 TAGT-7 1.32E08 4928 TAGT-7 3.26E08 5012 TAGT-8 1.76E09 5012 TAGT-8M 2.03E09 5014 TAGT-8 2.43E09 5014 TAGT-8M 3.87E09 5016 TAGT-8 3.56E09 5016 TAGT-8M 2.84E09 5020 TAGT-8 8.78E10 5020 TAGT-8M 7.00E09 5022 TAGT-8 3.68E09 5022 TAGT-8M 3.03E09 5023 TAGT-8 9.46E10 5023 TAGT-8M 5.77E09 5024 TAGT-8 4.52E09 5024 TAGT-8M 3.48E09 5030 TAGT-8 7.03E10 5030 TAGT-8M 4.27E09 5037 TAGT-8 1.06E09 5037 TAGT-8M 4.36E09 5039 TAGT-8 4.30E10 5039 TAGT-8M 2.69E09 5040 TAGT-8 4.37E10 5040 TAGT-8M 3.13E09 5041 TAGT-8 1.68E09 5041 TAGT-8M 1.67E09 5045 TAGT-8 1.00E09 5045 TAGT-8M 3.91E09 5048 TAGT-8 5.10E10 5048 TAGT-8M 2.52E09 5066 TAGT-8 5.23E09 5066 TAGT-8M 9.99E09 5070 TAGT-8 1.34E09 5070 TAGT-8M 6.63E09 5074 TAGT-8 4.31E09 5074 TAGT-8M 2.98E09 5082 TAGT-8 4.79E09 5082 TAGT-8M 3.23E09 5113 TAGT-12 6.80E09 5114 TAGT-12 3.42E08 5116 TAGT-12 1.46E08 5119 TAGT-12 7.54E09 5121 TAGT-12 9.29E09 5123 TAGT-12 5.67E09 5125 TAGT-12 2.42E08 5128 TAGT-12 7.12E09 5138 TAGT-12 8.55E09 5273 TAGT-1 1.34E08 5423 TAGT-9 4.90E09 5720 TAGT-11 1.93E08 5924 TAGT-12 5.95E08 5934 TAGT-12 1.66E08 6026 TAGT-2 2.95E09 6526 TAGT-4 1.16E08 7040 TAGT-8 2.72E08 7228 TAGT-12 7.62E09 7244 TAGT-12 1.05E08 7254 TAGT-12 1.07E08 7258 TAGT-12 9.72E09 7358 TAGT-3 5.15E08 7442 TAGT-9 6.83E09 7443 TAGT-9 1.27E08 HVR-H1_2 and 4052 TAGT-10 9.73E09 HVR-H2_1 4059 TAGT-10 3.30E07 5094 TAGT-10 4.34E08 5095 TAGT-10 1.27E08 5097 TAGT-10 1.27E08 5099 TAGT-10 4.20E08 5109 TAGT-10 2.59E08 5215 TAGT-1 6.64E09 5271 TAGT-1 1.24E08 5274 TAGT-1 2.52E08 5299 TAGT-6 1.37E08 5432 TAGT-9 4.83E09 5491 TAGT-11 1.43E08 5744 TAGT-11 1.14E08 5936 TAGT-10 1.75E08 6475 TAGT-4 7.22E09 7207 TAGT-5 4.99E10 7272 TAGT-5 3.49E09 7313 TAGT-5 5.69E09 7388 TAGT-5 2.72E09 7389 TAGT-5 4.50E09 7395 TAGT-5 1.65E08 7421 TAGT-9 2.47E08 7440 TAGT-9 6.79E09 7513 TAGT-9 8.43E09 HVR-H1_2 and 4812 TAGT-12 2.89E09 HVR-H2_2 4815 TAGT-12 5.91E09 4817 TAGT-12 2.06E09 4818 TAGT-12 1.02E09 4836 TAGT-12 2.49E09 4841 TAGT-12 4.50E10 4846 TAGT-12 3.19E09 4852 TAGT-12 2.26E09 4860 TAGT-12 2.44E09 4876 TAGT-12 7.75E09 4880 TAGT-12 2.77E09 4897 TAGT-12 6.83E10 4901 TAGT-12 3.19E09 4904 TAGT-12 5.39E09 5115 TAGT-12 1.16E08 5220 TAGT-1 5.03E09 5404 TAGT-6 3.30E09 5421 TAGT-9 1.05E08 5422 TAGT-9 5.12E09 5584 TAGT-2 1.76E09 5658 TAGT-11 2.61E10 7273 TAGT-5 6.01E09 7316 TAGT-5 2.04E08 7394 TAGT-5 8.75E09 HVR-H1_2 and 4037 TAGT-8 5.53E09 HVR-H2_3 4041 TAGT-8 1.54E09 4180 TAGT-10 7.39E08 4809 TAGT-12 3.69E10 4820 TAGT-12 3.96E09 4825 TAGT-12 6.05E09 4837 TAGT-12 5.36E09 4838 TAGT-12 2.52E09 4839 TAGT-12 6.16E09 4844 TAGT-12 6.95E10 4847 TAGT-12 3.64E10 4879 TAGT-12 3.13E09 4911 TAGT-7 1.50E08 5228 TAGT-1 3.06E08 5292 TAGT-1 1.57E08 5398 TAGT-9 1.97E08 7248 TAGT-12 1.28E08 7249 TAGT-12 5.36E09 7380 TAGT-5 1.24E08 7386 TAGT-5 1.32E08 7444 TAGT-9 5.53E09 7508 TAGT-9 1.36E08 HVR-H1_1 and 4097 TAGT-8 6.24E09 HVR-H2_1 5202 TAGT-1 1.50E08 5203 TAGT-1 1.31E08 5207 TAGT-1 7.44E09 5221 TAGT-1 1.18E08 5226 TAGT-1 8.36E09 5230 TAGT-1 9.21E09 5238 TAGT-1 5.04E08 5280 TAGT-1 8.43E09 5281 TAGT-1 4.70E09 5285 TAGT-1 1.42E08 5288 TAGT-1 1.08E08 5425 TAGT-9 2.15E08 7032 TAGT-8 2.08E08 7268 TAGT-5 3.76E09 7277 TAGT-5 2.56E09 7278 TAGT-5 1.53E08 7390 TAGT-5 1.44E09 HVR-H1_2 and 4102 TAGT-8 2.54E09 HVR-H2_5 4116 TAGT-10 <1.0E12 4827 TAGT-12 1.51E09 4834 TAGT-12 9.68E10 4851 TAGT-12 3.84E10 4863 TAGT-12 6.63E10 4875 TAGT-12 1.03E09 5217 TAGT-1 1.08E08 5921 TAGT-12 8.01E09 5930 TAGT-12 5.66E09 5932 TAGT-12 1.12E08 5968 TAGT-12 1.27E08 5980 TAGT-12 1.14E08 5990 TAGT-12 1.15E08 6010 TAGT-12 2.83E08 7310 TAGT-5 1.41E08 7379 TAGT-5 5.43E09 HVR-H1_1 and 4161 TAGT-8 2.98E08 HVR-H2_3 4177 TAGT-8 1.48E08 4823 TAGT-12 2.62E09 5192 TAGT-1 2.16E08 5193 TAGT-1 3.69E08 5204 TAGT-1 1.48E08 5234 TAGT-1 1.28E08 5237 TAGT-1 3.28E09 5615 TAGT-2 1.22E08 5733 TAGT-11 7.15E09 5741 TAGT-11 1.91E08 7324 TAGT-5 5.68E09 7367 TAGT-5 2.04E08 7372 TAGT-5 7.27E10 7506 TAGT-9 7.73E09 HVR-H1_3 and 5208 TAGT-1 3.36E09 HVR-H2_1 5283 TAGT-1 2.88E08 5303 TAGT-6 5.12E09 5310 TAGT-6 5.72E09 5314 TAGT-6 8.39E09 5318 TAGT-6 1.90E08 5342 TAGT-6 3.89E08 5359 TAGT-6 7.10E10 5365 TAGT-6 2.56E09 5370 TAGT-6 1.91E09 5413 TAGT-6 9.93E10 7275 TAGT-5 6.85E09 HVR-H1_1 and 4840 TAGT-12 2.08E09 HVR-H2_2 5195 TAGT-1 2.62E08 5201 TAGT-1 5.33E09 5211 TAGT-1 2.11E09 5216 TAGT-1 3.08E09 5286 TAGT-1 6.34E09 5287 TAGT-1 1.02E08 5290 TAGT-1 6.73E09 5722 TAGT-11 3.08E08 6030 TAGT-2 8.27E08 7370 TAGT-5 1.07E08 7385 TAGT-5 3.26E09 HVR-H1_3 and 4036 TAGT-8 3.13E09 HVR-H2_2 4096 TAGT-8 2.70E09 5323 TAGT-6 1.04E08 5387 TAGT-8 1.13E09 5756 TAGT-11 3.00E08 5985 TAGT-12 3.92E08 5986 TAGT-12 4.65E08 7163 TAGT-6 1.26E08 7375 TAGT-5 6.03E09 7391 TAGT-5 1.35E08 HVR-H1_3 and 4026 TAGT-8 3.08E09 HVR-H2_3 4858 TAGT-12 5.86E09 6533 TAGT-3 2.62E08 7159 TAGT-6 3.79E08 7166 TAGT-6 1.24E08 7239 TAGT-12 2.40E08 7274 TAGT-5 1.63E08 7433 TAGT-9 1.67E08 HVR-H1_3 and 4857 TAGT-12 4.05E09 HVR-H2_6 5227 TAGT-1 1.04E08 7221 TAGT-12 5.58E09 7229 TAGT-12 8.91E09 HVR-H1_2 and 4220 TAGT-6 5.72E08 HVR-H2_7 4861 TAGT-12 5.11E09 5284 TAGT-1 1.84E08 HVR-H1_1 and 4079 TAGT-6 3.15E08 HVR-H2_7 7129 TAGT-6 1.90E08 HVR-H1_3 and 4072 TAGT-6 6.95E09 HVR-H2_5 HVR-H1_3 and 5333 TAGT-6 5.02E09 HVR-H2_7

[0197] Hits containing the same HVR-H1 and HVR-H2 sequences were discovered that could bind different target antigens when these HVR-H1 and 2 sequences were paired with different HVR-H3 and VL sequences. For example, Hit IDs 4029, 7097, and 5906 contained the same HVR-H1 and HVR-H2 combination (HVR-H1 2 and HVR-H2 4) but were paired with different HVR-H3 and VL sequences, and bound three different target antigens (TAGT-8, TAGT-6, and TAGT-12, respectively). Hits 7040 and 5924 contained the same HVR-H1 and HVR-H2 combination (HVR-H1_2 and HVR-H2_6) but were paired with different HVR-H3 and VL sequences, and bound two different target antigens (TAGT-8 and TAGT-12, respectively).

[0198] Table 5 below shows sequence usage and number of targets bound for the HVR-H1 and HVR-H2s identified during the library analyses. Without wishing to be bound by theory, it is thought that a high number of antigens bound by an antibody comprising a given hypervariable region may be indicative of a high degree of flexibility of that particular hypervariable region, while a high segment usage of a given hypervariable region may be indicative of robust folding of the hypervariable region (and surrounding polypeptide sequence).

TABLE-US-00007 TABLE 5 target binding capability of HVR-H1 and HVR-H2 designed variants Sequence Number of Antigens Variant ID Usage Percent hit out of 14 HVR-H1_1 45.0% 11 HVR-H1_2 33.8% 14 HVR-H1_3 19.1% 8 HVR-H2_1 7.9% 8 HVR-H2_2 6.6% 8 HVR-H2_3 6.8% 11 HVR-H2_4 36.4% 12 HVR-H2_5 16.8% 8 HVR-H2_6 21.8% 13 HVR-H2_7 0.9% 3

[0199] Table 6 below shows sequence usage and number of antigens bound for the HVR-H1 and HVR-H2 combinations identified during the library analyses.

TABLE-US-00008 TABLE 6 HVR-H1 and HVR-H2 designed variants combination usage Sequence Number of Preference HVR-H1 HVR-H2 Usage Antigens hit Ranking Variant ID Variant ID Percent out of 14 Tier 1 HVR-H1_2 HVR-H2_6 7.6% 11 Tier 1 HVR-H1_2 HVR-H2_4 11.6% 10 Tier 1 HVR-H1_1 HVR-H2_4 11.2% 9 Tier 1 HVR-H1_1 HVR-H2_6 13.5% 7 Tier 1 HVR-H1_2 HVR-H2_1 3.6% 7 Tier 1 HVR-H1_2 HVR-H2_2 3.4% 7 Tier 1 HVR-H1_2 HVR-H2_3 3.4% 7 Tier 1 HVR-H1_1 HVR-H2_3 2.2% 7 Tier 1 HVR-H1_3 HVR-H2_3 1.1% 6 Tier 1 HVR-H1_3 HVR-H2_4 13.1% 5 Tier 1 HVR-H1_2 HVR-H2_5 2.4% 5 Tier 1 HVR-H1_1 HVR-H2_2 1.7% 5 Tier 1 HVR-H1_3 HVR-H2_2 1.4% 5 Tier 1 HVR-H1_1 HVR-H2_5 13.4% 4 Tier 2 HVR-H1_1 HVR-H2_1 2.6% 4 Tier 2 HVR-H1_3 HVR-H2_3 1.7% 3 Tier 2 HVR-H1_2 HVR-H2_7 0.4% 3 Tier 2 HVR-H1_3 HVR-H2_6 0.6% 2 Tier 3 HVR-H1_1 HVR-H2_7 0.3% 1 Tier 3 HVR-H1_3 HVR-H2_5 0.1% 1 Tier 3 HVR-H1_3 HVR-H2_7 0.1% 1

[0200] 74 HVR-H1 sequences (SEQ ID NOS: 1-52 and 137-158, Table 1) and 90 HVR-2 sequences (SEQ ID NOS: 53-136 and 159-164, Table 1) were identified that appeared in >1 of the unique antibody hits described above. When combined with various HVR-H3s and variable light chain domains, these HVRs were capable of forming antibodies that bound to multiple antigens. An additional 65 novel HVR-H1 and HVR-H2 sequence combinations were identified that appeared in >1 of the unique antibody hits described. Table 7 below shows HVR-H1 and HVR-H2 usage and number of antigens bound during the library analysis using these new HVR sequences.

TABLE-US-00009 TABLE 7 Usage of new HVR-H1 and HVR-H2 sequences Number of Antigens SEQ ID NO: Number of hits hit out of 14 1 12 8 5 10 7 16 9 6 8 37 5 22 12 5 21 7 5 31 14 4 12 12 4 4 11 4 7 11 4 26 7 4 19 6 4 23 6 4 47 6 4 18 5 4 24 5 4 28 5 4 9 5 4 38 4 4 49 4 4 25 16 3 50 13 3 51 8 3 27 5 3 11 5 3 40 4 3 43 4 3 20 3 3 33 3 3 42 3 3 45 3 3 13 27 2 34 7 2 35 5 2 41 5 2 3 4 2 15 3 2 30 3 2 44 3 2 46 3 2 32 2 2 37 2 2 39 2 2 2 2 2 14 2 2 48 6 1 29 3 1 6 3 1 17 2 1 36 2 1 52 2 1 10 2 1 63 40 7 93 12 5 66 8 5 122 7 5 65 6 5 105 5 5 124 14 4 123 7 4 70 4 4 110 46 3 129 26 3 121 15 3 89 9 3 134 9 3 128 7 3 60 4 3 67 4 3 95 3 3 117 14 2 82 11 2 130 11 2 132 10 2 53 9 2 131 7 2 109 6 2 72 5 2 118 5 2 100 4 2 103 4 2 106 4 2 61 3 2 71 3 2 75 3 2 77 3 2 79 3 2 108 3 2 112 3 2 113 3 2 55 2 2 56 2 2 59 2 2 62 2 2 64 2 2 68 2 2 69 2 2 73 2 2 74 2 2 76 2 2 78 2 2 81 2 2 83 2 2 86 2 2 90 2 2 91 2 2 99 2 2 107 2 2 135 2 2 136 2 2 126 29 1 116 10 1 87 5 1 84 4 1 85 4 1 92 4 1 104 4 1 57 3 1 80 3 1 94 3 1 96 3 1 101 3 1 111 3 1 114 3 1 120 3 1 133 3 1 54 2 1 58 2 1 88 2 1 97 2 1 98 2 1 102 2 1 115 2 1 119 2 1 125 2 1 127 2 1

[0201] Table 8 below shows usage and number of antigens bound for the combination of new HVR-H1 and HVR-H2 sequences.

TABLE-US-00010 TABLE 8 new HVR-H1 and HVR-H2 combination usage Preference HVR-H1 HVR-H2 Number of Number of Antigens Ranking SEQ ID NO: SEQ ID NO: hits hit out of 14 Tier 1 157 63 4 3 Tier 1 1 122 4 3 Tier 1 138 63 3 3 Tier 1 154 63 5 2 Tier 1 158 161 5 2 Tier 1 158 63 3 2 Tier 1 145 128 3 2 Tier 1 22 61 2 2 Tier 1 31 63 2 2 Tier 1 153 63 2 2 Tier 1 155 67 2 2 Tier 1 156 100 2 2 Tier 1 51 162 2 2 Tier 1 138 123 2 2 Tier 1 139 110 38 1 Tier 1 8 126 29 1 Tier 1 13 129 21 1 Tier 1 31 124 11 1 Tier 1 25 130 10 1 Tier 1 150 132 9 1 Tier 1 158 162 8 1 Tier 1 12 82 8 1 Tier 1 149 117 7 1 Tier 1 7 134 6 1 Tier 2 26 53 4 1 Tier 2 151 53 4 1 Tier 2 34 63 3 1 Tier 2 50 162 3 1 Tier 2 158 104 3 1 Tier 2 5 121 3 1 Tier 2 6 116 3 1 Tier 2 7 121 3 1 Tier 2 17 63 2 1 Tier 2 25 101 2 1 Tier 2 25 114 2 1 Tier 2 29 112 2 1 Tier 2 152 63 2 1 Tier 2 156 89 2 1 Tier 2 157 94 2 1 Tier 2 48 58 2 1 Tier 2 50 89 2 1 Tier 2 50 163 2 1 Tier 2 158 160 2 1 Tier 2 158 87 2 1 Tier 2 158 92 2 1 Tier 2 158 93 2 1 Tier 2 158 97 2 1 Tier 2 158 103 2 1 Tier 2 158 164 2 1 Tier 2 137 54 2 1 Tier 2 3 127 2 1 Tier 2 4 85 2 1 Tier 2 4 110 2 1 Tier 2 139 109 2 1 Tier 2 139 121 2 1 Tier 2 8 120 2 1 Tier 2 140 131 2 1 Tier 2 141 116 2 1 Tier 2 142 159 2 1 Tier 2 143 116 2 1 Tier 2 144 121 2 1 Tier 2 146 110 2 1 Tier 2 147 133 2 1 Tier 2 148 63 2 1 Tier 2 13 118 2 1

[0202] Table 9 shows affinity data for unique hits using the indicated new HVR-H1 and HVR-H2 sequences.

TABLE-US-00011 TABLE 9 Affinity data for confirmed hits using new HVR-H1 and HVR-H2 sequences HVR SEQ ID NO(S): Hit ID Antigen Kd (M) 8 4025 TAGT-8 2.89E09 8 4033 TAGT-8 8.75E10 8 4614 TAGT-8 3.53E10 8 4615 TAGT-8 2.28E10 8 4617 TAGT-8 2.88E10 8 4622 TAGT-8 2.74E10 8 4627 TAGT-8 1.82E10 8 4631 TAGT-8 1.83E10 8 4633 TAGT-8 3.22E10 8 4634 TAGT-8 2.07E10 8 4638 TAGT-8 3.14E10 8 4642 TAGT-8 1.89E10 8 4644 TAGT-8 2.48E10 8 4645 TAGT-8 2.96E10 8 4650 TAGT-8 3.57E10 8 4651 TAGT-8 3.01E10 8 4652 TAGT-8 2.94E10 8 4654 TAGT-8 2.32E10 8 4658 TAGT-8 1.42E10 8 4665 TAGT-8 3.69E10 8 4673 TAGT-8 3.23E10 8 4674 TAGT-8 5.02E10 8 4681 TAGT-8 5.43E10 8 4689 TAGT-8 1.63E10 8 4690 TAGT-8 4.67E10 8 5532 TAGT-2 3.65E09 8 5558 TAGT-2 2.43E09 8 5970 TAGT-12 1.35E08 8 6190 TAGT-10 2.55E09 8 6203 TAGT-10 1.05E08 8 7032 TAGT-8 2.08E08 8 7043 TAGT-8 1.34E08 8 7367 TAGT-5 2.04E08 8 BH3002 TAGT-8 2.51E10 8 BH3004 TAGT-8 3.00E10 8 BH3005 TAGT-8 3.46E10 8 BH3006 TAGT-8 1.94E10 13 4043 TAGT-8 2.69E09 13 4084 TAGT-8 2.94E09 13 4618 TAGT-8 1.08E09 13 4620 TAGT-8 3.48E10 13 4623 TAGT-8 4.85E10 13 4624 TAGT-8 1.00E12 13 4625 TAGT-8 4.02E10 13 4630 TAGT-8 2.67E10 13 4653 TAGT-8 3.27E10 13 4659 TAGT-8 2.12E10 13 4662 TAGT-8 8.98E10 13 4666 TAGT-8 1.17E09 13 4668 TAGT-8 5.79E10 13 4670 TAGT-8 8.21E10 13 4675 TAGT-8 1.00E12 13 4676 TAGT-8 1.62E10 13 4678 TAGT-8 5.98E10 13 4683 TAGT-8 8.97E10 13 4684 TAGT-8 6.69E10 13 4685 TAGT-8 4.78E10 13 4686 TAGT-8 4.78E10 13 4687 TAGT-8 4.08E10 13 5739 TAGT-11 1.29E08 13 7025 TAGT-8 4.87E08 13 7035 TAGT-8 3.04E09 13 7037 TAGT-8 2.10E08 13 7038 TAGT-8 2.33E08 25 4201 TAGT-6 1.41E08 25 4217 TAGT-6 9.67E08 25 4218 TAGT-6 2.85E08 25 4813 TAGT-12 2.45E10 25 5113 TAGT-12 6.80E09 25 5114 TAGT-12 3.42E08 25 5116 TAGT-12 1.46E08 25 5119 TAGT-12 7.54E09 25 5121 TAGT-12 9.29E09 25 5123 TAGT-12 5.67E09 25 5125 TAGT-12 2.42E08 25 5128 TAGT-12 7.12E09 25 5138 TAGT-12 8.55E09 25 5968 TAGT-12 1.27E08 25 5990 TAGT-12 1.15E08 25 7442 TAGT-9 6.83E09 31 4027 TAGT-8 1.55E09 31 4027 TAGT-8M 3.81E09 31 5020 TAGT-8 8.78E10 31 5020 TAGT-8M 7.00E09 31 5023 TAGT-8 9.46E10 31 5023 TAGT-8M 5.77E09 31 5030 TAGT-8 7.03E10 31 5030 TAGT-8M 4.27E09 31 5037 TAGT-8 1.06E09 31 5037 TAGT-8M 4.36E09 31 5039 TAGT-8 4.30E10 31 5039 TAGT-8M 2.69E09 31 5040 TAGT-8 4.37E10 31 5040 TAGT-8M 3.13E09 31 5045 TAGT-8 1.00E09 31 5045 TAGT-8M 3.91E09 31 5048 TAGT-8 5.10E10 31 5048 TAGT-8M 2.52E09 31 5066 TAGT-8 5.23E09 31 5066 TAGT-8M 9.99E09 31 5070 TAGT-8 1.34E09 31 5070 TAGT-8M 6.63E09 31 5658 TAGT-11 2.61E10 31 5926 TAGT-12 3.23E08 31 7394 TAGT-5 8.75E09 50 5929 TAGT-12 3.20E08 50 5978 TAGT-12 3.25E08 50 5999 TAGT-12 6.29E08 50 7077 TAGT-6 1.88E08 50 7090 TAGT-6 5.55E08 50 7128 TAGT-6 3.97E08 50 7209 TAGT-12 9.36E09 50 7219 TAGT-12 1.44E08 50 7235 TAGT-12 2.18E08 50 7240 TAGT-12 1.17E08 50 7256 TAGT-12 7.08E09 50 7257 TAGT-12 1.11E08 50 7375 TAGT-5 6.03E09 22 4116 TAGT-10 <1.0E12 22 4129 TAGT-6 <1.0E12 22 4140 TAGT-6 <1.0E12 22 4842 TAGT-12 4.01E10 22 5212 TAGT-1 9.19E09 22 5218 TAGT-1 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134 7231 TAGT-12 3.38E09 66 4161 TAGT-8 2.98E08 66 4180 TAGT-10 7.39E08 66 4809 TAGT-12 3.69E10 66 4847 TAGT-12 3.64E10 66 4879 TAGT-12 3.13E09 66 6533 TAGT-3 2.62E08 66 7372 TAGT-5 7.27E10 66 7386 TAGT-5 1.32E08 122 3757 TAGT-6 1.84E08 122 3869 TAGT-11 2.35E08 122 4163 TAGT-8 1.37E08 122 4828 TAGT-12 1.10E09 122 5103 TAGT-10 2.67E09 122 5163 TAGT-11 1.71E08 122 5740 TAGT-11 7.26E09 123 3762 TAGT-6 3.04E08 123 3780 TAGT-8 1.47E09 123 3865 TAGT-11 9.48E09 123 7030 TAGT-8 3.47E08 123 7035 TAGT-8 3.04E09 123 7055 TAGT-8 7.57E10 123 7358 TAGT-3 5.15E08 128 4101 TAGT-8 2.12E09 128 4661 TAGT-8 1.62E09 128 4792 TAGT-10 7.39E09 128 5997 TAGT-12 8.51E09 128 7040 TAGT-8 2.72E08 128 7221 TAGT-12 5.58E09 128 7228 TAGT-12 7.62E09 131 4103 TAGT-8 3.59E10 131 7215 TAGT-12 1.61E08 131 7229 TAGT-12 8.91E09 131 7243 TAGT-12 4.95E09 131 7244 TAGT-12 1.05E08 131 7254 TAGT-12 1.07E08 131 7258 TAGT-12 9.72E09 65 4037 TAGT-8 5.53E09 65 4823 TAGT-12 2.62E09 65 5292 TAGT-1 1.57E08 65 5741 TAGT-11 1.91E08 65 7239 TAGT-12 2.40E08 65 7433 TAGT-9 1.67E08 109 6179 TAGT-10 1.99E09 109 6184 TAGT-10 <1.0E12 109 6188 TAGT-10 8.76E09 109 6189 TAGT-10 2.38E10 109 6216 TAGT-10 6.58E10 109 6539 TAGT-4 3.45E09 72 5301 TAGT-6 2.61E07 72 5326 TAGT-6 7.84E07 72 5420 TAGT-6 1.41E08 72 5710 TAGT-11 1.01E08 72 5746 TAGT-11 5.00E09 87 4216 TAGT-6 2.59E08 87 5320 TAGT-6 6.13E07 87 5408 TAGT-6 2.36E08 87 7183 TAGT-6 1.48E08 87 7201 TAGT-6 3.26E08 105 5218 TAGT-1 6.04E09 105 5316 TAGT-6 1.14E08 105 5513 TAGT-2 1.07E09 105 6543 TAGT-3 6.78E08 105 7427 TAGT-9 5.58E09 118 4851 TAGT-12 3.84E10 118 7025 TAGT-8 4.87E08 118 7036 TAGT-8 1.59E08 118 7037 TAGT-8 2.10E08 118 7047 TAGT-8 2.15E08 60 5226 TAGT-1 8.36E09 60 5281 TAGT-1 4.70E09 60 5425 TAGT-9 2.15E08 60 5744 TAGT-11 1.14E08 67 4026 TAGT-8 3.08E09 67 4820 TAGT-12 3.96E09 67 4839 TAGT-12 6.16E09 67 7274 TAGT-5 1.63E08 70 4041 TAGT-8 1.54E09 70 4844 TAGT-12 6.95E10 70 7159 TAGT-6 3.79E08 70 7380 TAGT-5 1.24E08 84 7204 TAGT-5 2.33E09 84 7323 TAGT-5 3.23E09 84 7373 TAGT-5 5.13E09 84 7378 TAGT-5 5.66E09 85 7260 TAGT-5 2.30E09 85 7365 TAGT-5 1.82E09 85 7369 TAGT-5 2.46E09 85 7374 TAGT-5 1.97E08 92 4073 TAGT-6 <1.0E12 92 5355 TAGT-6 8.71E10 92 7080 TAGT-6 2.44E08 92 7081 TAGT-6 4.31E08 100 5328 TAGT-6 3.42E07 100 5417 TAGT-6 4.04E08 100 5974 TAGT-12 5.02E08 100 5977 TAGT-12 2.70E08 103 4075 TAGT-6 <1.0E12 103 5961 TAGT-12 2.41E08 103 5993 TAGT-12 1.13E08 103 7255 TAGT-12 1.20E08 104 5912 TAGT-12 1.68E08 104 5923 TAGT-12 1.60E08 104 5978 TAGT-12 3.25E08 104 7226 TAGT-12 7.57E09 106 4141 TAGT-6 <1.0E12 106 4222 TAGT-6 5.55E08 106 5321 TAGT-6 7.16E07 106 7317 TAGT-5 2.02E08 57 5303 TAGT-6 5.12E09 57 5359 TAGT-6 7.10E10 57 5365 TAGT-6 2.56E09 61 5230 TAGT-1 9.21E09 61 5271 TAGT-1 1.24E08 61 7207 TAGT-5 4.99E10 71 5336 TAGT-6 6.04E07 71 5418 TAGT-6 2.02E08 71 5438 TAGT-9 9.30E09 75 5194 TAGT-1 1.29E08 75 5235 TAGT-1 1.41E08 75 5403 TAGT-6 8.26E09 77 4063 TAGT-10 1.90E08 77 4067 TAGT-10 1.97E08 77 7429 TAGT-9 2.12E08 79 5353 TAGT-6 1.61E08 79 7419 TAGT-9 1.72E08 79 7431 TAGT-9 3.53E08 80 7276 TAGT-5 1.02E08 80 7311 TAGT-5 9.20E09 80 7371 TAGT-5 2.31E08 94 5371 TAGT-6 3.97E09 94 7088 TAGT-6 4.36E08 94 7100 TAGT-6 3.50E08 95 5236 TAGT-1 1.49E08 95 5983 TAGT-12 2.09E08 95 7128 TAGT-6 3.97E08 96 7077 TAGT-6 1.88E08 96 7107 TAGT-6 1.22E07 96 7109 TAGT-6 3.20E08 10 3761 TAGT-6 9.65E08 10 4217 TAGT-6 9.67E08 10 4218 TAGT-6 2.85E08 108 4034 TAGT-8 4.27E09 108 5351 TAGT-6 7.29E09 108 5357 TAGT-6 7.14E09 111 4827 TAGT-12 1.51E09 111 4834 TAGT-12 9.68E10 111 4875 TAGT-12 1.03E09 112 4025 TAGT-8 2.89E09 112 5930 TAGT-12 5.66E09 112 5932 TAGT-12 1.12E08 113 4116 TAGT-10 <1.0E12 113 4863 TAGT-12 6.63E10 113 5980 TAGT-12 1.14E08 114 5921 TAGT-12 8.01E09 114 5968 TAGT-12 1.27E08 114 5990 TAGT-12 1.15E08 120 6180 TAGT-10 6.11E09 120 6190 TAGT-10 2.55E09 120 6203 TAGT-10 1.05E08 133 5935 TAGT-12 8.78E09 133 6008 TAGT-12 5.10E08 133 7222 TAGT-12 1.26E09 54 7277 TAGT-5 2.56E09 54 7390 TAGT-5 1.44E09 55 5238 TAGT-1 5.04E08 55 5370 TAGT-6 1.91E09 56 5285 TAGT-1 1.42E08 56 5310 TAGT-6 5.72E09 58 5314 TAGT-6 8.39E09 58 5342 TAGT-6 3.89E08 59 5202 TAGT-1 1.50E08 59 7032 TAGT-8 2.08E08 62 5220 TAGT-1 5.03E09 62 7163 TAGT-6 1.26E08 64 5211 TAGT-1 2.11E09 64 5584 TAGT-2 1.76E09 68 4177 TAGT-8 1.48E08 68 5234 TAGT-1 1.28E08 69 4838 TAGT-12 2.52E09 69 7166 TAGT-6 1.24E08 73 4878 TAGT-12 4.07E09 73 5315 TAGT-6 2.10E06 74 3760 TAGT-6 1.26E08 76 5297 TAGT-6 1.77E06 76 5745 TAGT-11 1.06E08 78 4058 TAGT-10 1.13E08 78 5291 TAGT-1 6.57E09 81 5212 TAGT-1 9.19E09 81 5568 TAGT-2 1.14E09 83 5411 TAGT-6 1.25E08 83 5565 TAGT-2 3.02E09 86 4129 TAGT-6 <1.0E12 86 6473 TAGT-4 2.30E08 88 5905 TAGT-12 3.83E08 88 5919 TAGT-12 2.38E08 90 4029 TAGT-8 1.89E09 90 7097 TAGT-6 2.43E08 91 5272 TAGT-1 2.49E08 91 7242 TAGT-12 1.71E08 97 5915 TAGT-12 1.82E08 97 5964 TAGT-12 1.40E08 98 4131 TAGT-6 <1.0E12 98 5347 TAGT-6 1.21E08 99 7090 TAGT-6 5.55E08 102 6004 TAGT-12 5.50E08 102 7251 TAGT-12 2.69E08 107 4133 TAGT-6 3.90E10 107 7262 TAGT-5 2.63E09 115 7310 TAGT-5 1.41E08 115 7379 TAGT-5 5.43E09 119 6193 TAGT-10 3.18E09 119 6220 TAGT-10 3.45E09 125 4030 TAGT-8 4.90E09 125 7038 TAGT-8 2.33E08 127 7044 TAGT-8 1.12E09 127 7045 TAGT-8 1.11E09 135 4204 TAGT-10 6.83E09 135 4204 TAGT-10M 6.89E09 135 5423 TAGT-9 4.90E09 136 4861 TAGT-12 5.11E09 136 7129 TAGT-6 1.90E08 139 and 110 4181 TAGT-10 4.27E08 139 and 110 4693 TAGT-10 4.87E10 139 and 110 4696 TAGT-10 4.58E10 139 and 110 4697 TAGT-10 6.21E10 139 and 110 4698 TAGT-10 5.70E10 139 and 110 4700 TAGT-10 2.62E10 139 and 110 4701 TAGT-10 5.60E10 139 and 110 4702 TAGT-10 5.02E10 139 and 110 4703 TAGT-10 2.85E10 139 and 110 4704 TAGT-10 6.65E10 139 and 110 4705 TAGT-10 3.02E10 139 and 110 4706 TAGT-10 2.50E10 139 and 110 4707 TAGT-10 4.29E10 139 and 110 4708 TAGT-10 5.29E10 139 and 110 4710 TAGT-10 6.26E10 139 and 110 4714 TAGT-10 4.46E10 139 and 110 4717 TAGT-10 4.61E10 139 and 110 4718 TAGT-10 5.32E10 139 and 110 4722 TAGT-10 7.46E10 139 and 110 4725 TAGT-10 4.84E10 139 and 110 4729 TAGT-10 8.80E10 139 and 110 4731 TAGT-10 4.67E10 139 and 110 4732 TAGT-10 3.33E10 139 and 110 4738 TAGT-10 5.34E10 139 and 110 4744 TAGT-10 3.73E10 139 and 110 4748 TAGT-10 3.92E10 139 and 110 4749 TAGT-10 2.55E10 139 and 110 4750 TAGT-10 7.86E10 139 and 110 4753 TAGT-10 3.43E10 139 and 110 4759 TAGT-10 6.59E10 139 and 110 4766 TAGT-10 4.09E10 139 and 110 4788 TAGT-10 2.88E10 139 and 110 4794 TAGT-10 5.56E10 139 and 110 4803 TAGT-10 1.88E10 139 and 110 4805 TAGT-10 4.26E10 139 and 110 4808 TAGT-10 8.28E10 139 and 110 4909 TAGT-10 2.90E10 139 and 110 6191 TAGT-10 6.58E11 8 and 126 4033 TAGT-8 8.75E10 8 and 126 4614 TAGT-8 3.53E10 8 and 126 4615 TAGT-8 2.28E10 8 and 126 4617 TAGT-8 2.88E10 8 and 126 4622 TAGT-8 2.74E10 8 and 126 4627 TAGT-8 1.82E10 8 and 126 4631 TAGT-8 1.83E10 8 and 126 4633 TAGT-8 3.22E10 8 and 126 4634 TAGT-8 2.07E10 8 and 126 4638 TAGT-8 3.14E10 8 and 126 4642 TAGT-8 1.89E10 8 and 126 4644 TAGT-8 2.48E10 8 and 126 4645 TAGT-8 2.96E10 8 and 126 4650 TAGT-8 3.57E10 8 and 126 4651 TAGT-8 3.01E10 8 and 126 4652 TAGT-8 2.94E10 8 and 126 4654 TAGT-8 2.32E10 8 and 126 4658 TAGT-8 1.42E10 8 and 126 4665 TAGT-8 3.69E10 8 and 126 4673 TAGT-8 3.23E10 8 and 126 4674 TAGT-8 5.02E10 8 and 126 4681 TAGT-8 5.43E10 8 and 126 4689 TAGT-8 1.63E10 8 and 126 4690 TAGT-8 4.67E10 8 and 126 7043 TAGT-8 1.34E08 8 and 126 BH3002 TAGT-8 2.51E10 8 and 126 BH3004 TAGT-8 3.00E10 8 and 126 BH3005 TAGT-8 3.46E10 8 and 126 BH3006 TAGT-8 1.94E10 13 and 129 4084 TAGT-8 2.94E09 13 and 129 4618 TAGT-8 1.08E09 13 and 129 4620 TAGT-8 3.48E10 13 and 129 4623 TAGT-8 4.85E10 13 and 129 4624 TAGT-8 1.00E12 13 and 129 4625 TAGT-8 4.02E10 13 and 129 4630 TAGT-8 2.67E10 13 and 129 4653 TAGT-8 3.27E10 13 and 129 4659 TAGT-8 2.12E10 13 and 129 4662 TAGT-8 8.98E10 13 and 129 4666 TAGT-8 1.17E09 13 and 129 4668 TAGT-8 5.79E10 13 and 129 4670 TAGT-8 8.21E10 13 and 129 4675 TAGT-8 1.00E12 13 and 129 4676 TAGT-8 1.62E10 13 and 129 4678 TAGT-8 5.98E10 13 and 129 4683 TAGT-8 8.97E10 13 and 129 4684 TAGT-8 6.69E10 13 and 129 4685 TAGT-8 4.78E10 13 and 129 4686 TAGT-8 4.78E10 13 and 129 4687 TAGT-8 4.08E10 31 and 124 4027 TAGT-8 1.55E09 31 and 124 4027 TAGT-8M 3.81E09 31 and 124 5020 TAGT-8 8.78E10 31 and 124 5020 TAGT-8M 7.00E09 31 and 124 5023 TAGT-8 9.46E10 31 and 124 5023 TAGT-8M 5.77E09 31 and 124 5030 TAGT-8 7.03E10 31 and 124 5030 TAGT-8M 4.27E09 31 and 124 5037 TAGT-8 1.06E09 31 and 124 5037 TAGT-8M 4.36E09 31 and 124 5039 TAGT-8 4.30E10 31 and 124 5039 TAGT-8M 2.69E09 31 and 124 5040 TAGT-8 4.37E10 31 and 124 5040 TAGT-8M 3.13E09 31 and 124 5045 TAGT-8 1.00E09 31 and 124 5045 TAGT-8M 3.91E09 31 and 124 5048 TAGT-8 5.10E10 31 and 124 5048 TAGT-8M 2.52E09 31 and 124 5066 TAGT-8 5.23E09 31 and 124 5066 TAGT-8M 9.99E09 31 and 124 5070 TAGT-8 1.34E09 31 and 124 5070 TAGT-8M 6.63E09 25 and 130 4813 TAGT-12 2.45E10 25 and 130 5113 TAGT-12 6.80E09 25 and 130 5114 TAGT-12 3.42E08 25 and 130 5116 TAGT-12 1.46E08 25 and 130 5119 TAGT-12 7.54E09 25 and 130 5121 TAGT-12 9.29E09 25 and 130 5123 TAGT-12 5.67E09 25 and 130 5125 TAGT-12 2.42E08 25 and 130 5128 TAGT-12 7.12E09 25 and 130 5138 TAGT-12 8.55E09 150 and 132 4032 TAGT-8 5.11E09 150 and 132 4032 TAGT-8M 4.84E09 150 and 132 5012 TAGT-8 1.76E09 150 and 132 5012 TAGT-8M 2.03E09 150 and 132 5014 TAGT-8 2.43E09 150 and 132 5014 TAGT-8M 3.87E09 150 and 132 5016 TAGT-8 3.56E09 150 and 132 5016 TAGT-8M 2.84E09 150 and 132 5022 TAGT-8 3.68E09 150 and 132 5022 TAGT-8M 3.03E09 150 and 132 5024 TAGT-8 4.52E09 150 and 132 5024 TAGT-8M 3.48E09 150 and 132 5041 TAGT-8 1.68E09 150 and 132 5041 TAGT-8M 1.67E09 150 and 132 5074 TAGT-8 4.31E09 150 and 132 5074 TAGT-8M 2.98E09 150 and 132 5082 TAGT-8 4.79E09 150 and 132 5082 TAGT-8M 3.23E09 158 and 162 5962 TAGT-12 8.06E08 158 and 162 5996 TAGT-12 2.21E08 158 and 162 6000 TAGT-12 7.86E08 158 and 162 7210 TAGT-12 9.85E09 158 and 162 7218 TAGT-12 1.49E08 158 and 162 7225 TAGT-12 9.53E09 158 and 162 7241 TAGT-12 6.43E09 158 and 162 7247 TAGT-12 8.93E09 12 and 82 4048 TAGT-10 3.24E09 12 and 82 4723 TAGT-10 9.11E10 12 and 82 4733 TAGT-10 3.05E10 12 and 82 4734 TAGT-10 5.72E10 12 and 82 4767 TAGT-10 2.77E10 12 and 82 4771 TAGT-10 7.23E10 12 and 82 4797 TAGT-10 5.63E10 12 and 82 4807 TAGT-10 1.17E09 149 and 117 4031 TAGT-8 1.06E09 149 and 117 5126 TAGT-8 9.54E09 149 and 117 5129 TAGT-8 1.12E09 149 and 117 5132 TAGT-8 3.06E09 149 and 117 5145 TAGT-8 7.00E09 149 and 117 7068 TAGT-8 1.11E08 149 and 117 7073 TAGT-8 3.19E09 7 and 134 3898 TAGT-11 1.83E08 7 and 134 5149 TAGT-11 2.91E09 7 and 134 5159 TAGT-11 4.09E09 7 and 134 5160 TAGT-11 8.07E09 7 and 134 5162 TAGT-11 9.87E09 7 and 134 5165 TAGT-11 4.06E09 154 and 63 4812 TAGT-12 2.89E09 154 and 63 4904 TAGT-12 5.39E09 154 and 63 5115 TAGT-12 1.16E08 154 and 63 5421 TAGT-9 1.05E08 154 and 63 5422 TAGT-9 5.12E09 158 and 161 5922 TAGT-12 1.95E08 158 and 161 7135 TAGT-6 3.17E08 158 and 161 7245 TAGT-12 1.38E08 158 and 161 7246 TAGT-12 6.22E09 158 and 161 7252 TAGT-12 9.56E09 26 and 53 4052 TAGT-10 9.73E09 26 and 53 5094 TAGT-10 4.34E08 26 and 53 5097 TAGT-10 1.27E08 26 and 53 5109 TAGT-10 2.59E08 151 and 53 4059 TAGT-10 3.30E07 151 and 53 5095 TAGT-10 1.27E08 151 and 53 5099 TAGT-10 4.20E08 151 and 53 5936 TAGT-10 1.75E08 157 and 63 4036 TAGT-8 3.13E09 157 and 63 4096 TAGT-8 2.70E09 157 and 63 5323 TAGT-6 1.04E08 157 and 63 7391 TAGT-5 1.35E08 1 and 122 3757 TAGT-6 1.84E08 1 and 122 3869 TAGT-11 2.35E08 1 and 122 5103 TAGT-10 2.67E09 1 and 122 5163 TAGT-11 1.71E08 34 and 63 4836 TAGT-12 2.49E09 34 and 63 4852 TAGT-12 2.26E09 34 and 63 4876 TAGT-12 7.75E09 50 and 162 7240 TAGT-12 1.17E08 50 and 162 7256 TAGT-12 7.08E09 50 and 162 7257 TAGT-12 1.11E08 158 and 63 5387 TAGT-8 1.13E09 158 and 63 5985 TAGT-12 3.92E08 158 and 63 5986 TAGT-12 4.65E08 158 and 104 5912 TAGT-12 1.68E08 158 and 104 5923 TAGT-12 1.60E08 158 and 104 7226 TAGT-12 7.57E09 5 and 121 4060 TAGT-10 1.10E08 5 and 121 4798 TAGT-10 4.35E09 5 and 121 6219 TAGT-10 3.15E09 6 and 116 4752 TAGT-10 3.34E09 6 and 116 6210 TAGT-10 5.17E10 6 and 116 6212 TAGT-10 2.25E09 138 and 63 4840 TAGT-12 2.08E09 138 and 63 5722 TAGT-11 3.08E08 138 and 63 7385 TAGT-5 3.26E09 7 and 121 4065 TAGT-10 4.31E08 7 and 121 4182 TAGT-10 4.24E09 7 and 121 4741 TAGT-10 1.66E09 145 and 128 4101 TAGT-8 2.12E09 145 and 128 4661 TAGT-8 1.62E09 145 and 128 4792 TAGT-10 7.39E09 17 and 63 4818 TAGT-12 1.02E09 17 and 63 4841 TAGT-12 4.50E10 22 and 61 5271 TAGT-1 1.24E08 22 and 61 7207 TAGT-5 4.99E10 25 and 101 4217 TAGT-6 9.67E08 25 and 101 4218 TAGT-6 2.85E08 25 and 114 5968 TAGT-12 1.27E08 25 and 114 5990 TAGT-12 1.15E08 29 and 112 5930 TAGT-12 5.66E09 29 and 112 5932 TAGT-12 1.12E08 31 and 63 5658 TAGT-11 2.61E10 31 and 63 7394 TAGT-5 8.75E09 152 and 63 4897 TAGT-12 6.83E10 152 and 63 4901 TAGT-12 3.19E09 153 and 63 4817 TAGT-12 2.06E09 153 and 63 7316 TAGT-5 2.04E08 155 and 67 4026 TAGT-8 3.08E09 155 and 67 7274 TAGT-5 1.63E08 156 and 89 7079 TAGT-6 2.99E08 156 and 89 7133 TAGT-6 4.03E08 156 and 100 5417 TAGT-6 4.04E08 156 and 100 5974 TAGT-12 5.02E08 157 and 94 7088 TAGT-6 4.36E08 157 and 94 7100 TAGT-6 3.50E08 48 and 58 5314 TAGT-6 8.39E09 48 and 58 5342 TAGT-6 3.89E08 50 and 89 5929 TAGT-12 3.20E08 50 and 89 7219 TAGT-12 1.44E08 50 and 163 5999 TAGT-12 6.29E08 50 and 163 7235 TAGT-12 2.18E08 158 and 160 5911 TAGT-12 3.35E08 158 and 160 7216 TAGT-12 1.88E08 158 and 87 4216 TAGT-6 2.59E08 158 and 87 7201 TAGT-6 3.26E08 158 and 92 7080 TAGT-6 2.44E08 158 and 92 7081 TAGT-6 4.31E08 158 and 93 7078 TAGT-6 2.52E08 158 and 93 7087 TAGT-6 6.96E08 158 and 97 5915 TAGT-12 1.82E08 158 and 97 5964 TAGT-12 1.40E08 158 and 103 5961 TAGT-12 2.41E08 158 and 103 7255 TAGT-12 1.20E08 158 and 164 7211 TAGT-12 1.26E08 158 and 164 7220 TAGT-12 9.12E09 51 and 162 4074 TAGT-6 1.95E08 51 and 162 7237 TAGT-12 2.13E08 137 and 54 7277 TAGT-5 2.56E09 137 and 54 7390 TAGT-5 1.44E09 3 and 127 7044 TAGT-8 1.12E09 3 and 127 7045 TAGT-8 1.11E09 4 and 85 7260 TAGT-5 2.30E09 4 and 85 7374 TAGT-5 1.97E08 4 and 110 6183 TAGT-10 2.70E09 4 and 110 6206 TAGT-10 3.44E09 138 and 123 3762 TAGT-6 3.04E08 138 and 123 3865 TAGT-11 9.48E09 139 and 109 6184 TAGT-10 <1.0E12 139 and 109 6216 TAGT-10 6.58E10 139 and 121 6187 TAGT-10 2.74E08 139 and 121 6197 TAGT-10 8.56E09 8 and 120 6190 TAGT-10 2.55E09 8 and 120 6203 TAGT-10 1.05E08 140 and 131 7215 TAGT-12 1.61E08 140 and 131 7243 TAGT-12 4.95E09 141 and 116 6204 TAGT-10 6.46E09 141 and 116 6214 TAGT-10 1.51E09 142 and 159 5554 TAGT-2 2.88E09 142 and 159 5622 TAGT-2 3.06E09 143 and 116 6194 TAGT-10 2.49E10 143 and 116 6196 TAGT-10 <1.0E12 144 and 121 6185 TAGT-10 1.57E09 144 and 121 6209 TAGT-10 3.35E09 146 and 110 4055 TAGT-10 1.07E08 146 and 110 4743 TAGT-10 7.40E09 147 and 133 5935 TAGT-12 8.78E09 147 and 133 6008 TAGT-12 5.10E08 148 and 63 5195 TAGT-1 2.62E08 148 and 63 5290 TAGT-1 6.73E09 13 and 118 7025 TAGT-8 4.87E08 13 and 118 7037 TAGT-8 2.10E08

[0203] An HVR-H1 comprising SEQ ID NO:16 was used in 8 unique hits. Using this same HVR-H1 sequence, but different sequences of the other HVRs, those 8 hits were capable of binding to 5 different target antigens. Exemplary hit IDs 4034, 6010, and 7183, which bound to TAGT-8, TAGT-12, and TAGT-6, respectively, contained an HVR-H1 comprising SEQ ID NO:16.

[0204] An HVR-H2 comprising SEQ ID NO:63 was used in 40 unique hits. Using this same HVR-H2 sequence, but different sequences of the other HVRs, those 40 hits were capable of binding to 7 different target antigens. Exemplary hit IDs 4036, 5115, and 5404, which bound to TAGT-8, TAGT-12, and TAGT-6, respectively, contained an HVR-H2 comprising SEQ ID NO:63.

[0205] Exemplary hit IDs 3757 and 5103 contained the same heavy chain variable region, including the same HVR-H1 and HVR-H2 sequences (SEQ ID NOS: 1 and 122), but when combined with different variable light chain domains, they bound to two different target antigens (TAGT-6 and TAGT-10, respectively). Two additional hits with these same HVR-H1 and HVR-H2 sequences could bind to another target antigen, TAGT-11.

[0206] Exemplary hit ID 4027, containing the HVR-H1 and HVR-H2 sequences of SEQ ID NOS:31 and 124, was capable of binding the same antigen from two different species (TAGT-8H and TAGT-8M). Several other hits with these same HVR-H1 and HVR-H2 sequences demonstrated species cross-reactivity.

[0207] The novel methodology employed to identify the dynamic motif of the redefined hyper-variable regions of antibodies based upon structural and sequence variability has led to the design of a limited number of V.sub.H components that can bind to the same or multiple different targets depending upon the V.sub.L segment with which the V.sub.H components are paired. The data and antibodies described herein reveals that the heavy chain library, either used as a whole set or a subset, is robust enough to serve as the V.sub.H component for antibody discovery.

SEQUENCES

[0208] All polypeptide sequences are presented N-terminal to C-terminal unless otherwise noted. All polynucleotide sequences are presented 5 to 3 unless otherwise noted.

TABLE-US-00012 DesignedHVR-H1sequence1: (SEQIDNO:1) FTFTDYGIHWV DesignedHVR-H1sequence2: (SEQIDNO:2) FTFTGYAIHWV DesignedHVR-H1sequence3: (SEQIDNO:3) FTFTNYGIHWV DesignedHVR-H1sequence4: (SEQIDNO:4) YTFSDYAIHWV DesignedHVR-H1sequence5: (SEQIDNO:5) YTFSDYGIHWV DesignedHVR-H1sequence6: (SEQIDNO:6) YTFSGYAIHWV DesignedHVR-H1sequence7: (SEQIDNO:7) YTFSGYGIHWV DesignedHVR-H1sequence8: (SEQIDNO:8) YTFSNYGIHWV DesignedHVR-H1sequence9: (SEQIDNO:9) YTFSSYGIHWV DesignedHVR-H1sequence10: (SEQIDNO:10) YTFSGYWIHWV DesignedHVR-H1sequence11: (SEQIDNO:11) YTFSNYWIHWV DesignedHVR-H1sequence12: (SEQIDNO:12) FTFSGYWIHWV DesignedHVR-H1sequence13: (SEQIDNO:13) FTFSNYWIHWV DesignedHVR-H1sequence14: (SEQIDNO:14) YTFSDYWIHWV DesignedHVR-H1sequence15: (SEQIDNO:15) YSISSGHHWAWI DesignedHVR-H1sequence16: (SEQIDNO:16) YSISSGHYWNWI DesignedHVR-H1sequence17: (SEQIDNO:17) YSISSGHYWSWI DesignedHVR-H1sequence18: (SEQIDNO:18) YSISSGHYWTWI DesignedHVR-H1sequence19: (SEQIDNO:19) YSISSGYHWAWI DesignedHVR-H1sequence20: (SEQIDNO:20) YSISSGYHWDWI DesignedHVR-H1sequence21: (SEQIDNO:21) YSISSGYHWGWI DesignedHVR-H1sequence22: (SEQIDNO:22) YSISSGYHWNWI DesignedHVR-H1sequence23: (SEQIDNO:23) YSISSGYHWSWI DesignedHVR-H1sequence24: (SEQIDNO:24) YSISSGHHWDWI DesignedHVR-H1sequence25: (SEQIDNO:25) YSISSGYYWDWI DesignedHVR-H1sequence26: (SEQIDNO:26) YSISSGYYWNWI DesignedHVR-H1sequence27: (SEQIDNO:27) YSISSGYYWTWI DesignedHVR-H1sequence28: (SEQIDNO:28) YSITSGHHWAWI DesignedHVR-H1sequence29: (SEQIDNO:29) YSITSGHHWDWI DesignedHVR-H1sequence30: (SEQIDNO:30) YSITSGHHWGWI DesignedHVR-H1sequence31: (SEQIDNO:31) YSITSGHHWNWI DesignedHVR-H1sequence32: (SEQIDNO:32) YSITSGHHWSWI DesignedHVR-H1sequence33: (SEQIDNO:33) YSISSGHHWGWI DesignedHVR-H1sequence34: (SEQIDNO:34) YSITSGHYWAWI DesignedHVR-H1sequence35: (SEQIDNO:35) YSITSGHYWDWI DesignedHVR-H1sequence36: (SEQIDNO:36) YSITSGHYWGWI DesignedHVR-H1sequence37: (SEQIDNO:37) YSITSGHYWNWI DesignedHVR-H1sequence38: (SEQIDNO:38) YSITSGHYWSWI DesignedHVR-H1sequence39: (SEQIDNO:39) YSITSGYHWAWI DesignedHVR-H1sequence40: (SEQIDNO:40) YSITSGYHWGWI DesignedHVR-H1sequence41: (SEQIDNO:41) YSISSGHHWNWI DesignedHVR-H1sequence42: (SEQIDNO:42) YSITSGYHWNWI DesignedHVR-H1sequence43: (SEQIDNO:43) YSITSGYHWSWI DesignedHVR-H1sequence44: (SEQIDNO:44) YSITSGYYWDWI DesignedHVR-H1sequence45: (SEQIDNO:45) YSISSGHHWTWI DesignedHVR-H1sequence46: (SEQIDNO:46) YSISSGHYWDWI DesignedHVR-H1sequence47: (SEQIDNO:47) FSLSTSGVAVSWI DesignedHVR-H1sequence48: (SEQIDNO:48) FSLSTGGVAVGWI DesignedHVR-H1sequence49: (SEQIDNO:49) FSLSTGGVAVSWI DesignedHVR-H1sequence50: (SEQIDNO:50) FSLSTGGVGVAWI DesignedHVR-H1sequence51: (SEQIDNO:51) FSLSTGGVGVSWI DesignedHVR-H1sequence52: (SEQIDNO:52) FSLSTSGVAVAWI DesignedHVR-H1sequence53: (SEQIDNO:137) FTFSDYAIHWV DesignedHVR-H1sequence54: (SEQIDNO:138) FTFSDYGIHWV DesignedHVR-H1sequence55: (SEQIDNO:139) YTFSNYAIHWV DesignedHVR-H1sequence56: (SEQIDNO:140) YTFSSYAIHWV DesignedHVR-H1sequence57: (SEQIDNO:141) YTFTDYAIHWV DesignedHVR-H1sequence58: (SEQIDNO:142) YTFTDYGIHWV DesignedHVR-H1sequence59: (SEQIDNO:143) YTFTNYAIHWV DesignedHVR-H1sequence60: (SEQIDNO:144) YTFTNYGIHWV DesignedHVR-H1sequence61: (SEQIDNO:145) FTFSGYGIHWV DesignedHVR-H1sequence62: (SEQIDNO:146) FTFSNYAIHWV DesignedHVR-H1sequence63: (SEQIDNO:147) FTFSSYGIHWV DesignedHVR-H1sequence64: (SEQIDNO:148) FTFSDYWIHWV DesignedHVR-H1sequence65: (SEQIDNO:149) FTFTSYWIHWV DesignedHVR-H1sequence66: (SEQIDNO:150) YSISSGYYWGWI DesignedHVR-H1sequence67: (SEQIDNO:151) YSITSGYYWNWI DesignedHVR-H1sequence68: (SEQIDNO:152) YSITSGYYWSWI DesignedHVR-H1sequence69: (SEQIDNO:153) YSISSGHYWAWI DesignedHVR-H1sequence70: (SEQIDNO:154) YSISSGHYWGWI DesignedHVR-H1sequence71: (SEQIDNO:155) FSLSTSGVAVGWI DesignedHVR-H1sequence72: (SEQIDNO:156) FSLSTSGVGVAWI DesignedHVR-H1sequence73: (SEQIDNO:157) FSLSTSGVGVGWI DesignedHVR-H1sequence74: (SEQIDNO:158) FSLSTGGVGVGWI DesignedHVR-H2sequence1: (SEQIDNO:53) LARIDWDDDKRYSPSLKSRL DesignedHVR-H2sequence2: (SEQIDNO:54) LALIDWDDDKRYSPSLKSRL DesignedHVR-H2sequence3: (SEQIDNO:55) LALIDWDDDKRYSTSLKSRL DesignedHVR-H2sequence4: (SEQIDNO:56) LALIDWDDDKYYSPSLKSRL DesignedHVR-H2sequence5: (SEQIDNO:57) LALIDWADDKYYSPSLKSRL DesignedHVR-H2sequence6: (SEQIDNO:58) LALIDWAGDKSYSTSLKSRL DesignedHVR-H2sequence7: (SEQIDNO:59) LARIDWDDDKYYSPSLKSRL DesignedHVR-H2sequence8: (SEQIDNO:60) LARIDWDDDKYYSTSLKSRL DesignedHVR-H2sequence9: (SEQIDNO:61) LARIDWDGDKYYSTSLKSRL DesignedHVR-H2sequence10: (SEQIDNO:62) IGDIYHSGSTYYSPSLKSRV DesignedHVR-H2sequence11: (SEQIDNO:63) IGEIYHSGSTYYSPSLKSRV DesignedHVR-H2sequence12: (SEQIDNO:64) IGEIYYSGSTYYSPSLKSRV DesignedHVR-H2sequence13: (SEQIDNO:65) IGSIYHSGNTNYNPSLKSRV DesignedHVR-H2sequence14: (SEQIDNO:66) IGEIYHSGNTYYNPSLKSRV DesignedHVR-H2sequence15: (SEQIDNO:67) IGEIYHSGSTYYNPSLKSRV DesignedHVR-H2sequence16: (SEQIDNO:68) IGEIYYSGSTYYNPSLKSRV DesignedHVR-H2sequence17: (SEQIDNO:69) IGDIYHSGNTYYNPSLKSRV DesignedHVR-H2sequence18: (SEQIDNO:70) IGDIYHSGSTYYNPSLKSRV DesignedHVR-H2sequence19: (SEQIDNO:71) VSAISGYGDTTYYADSVKGRF DesignedHVR-H2sequence20: (SEQIDNO:72) VSAISGYGGSTYYADSVKGRF DesignedHVR-H2sequence21: (SEQIDNO:73) VSAISGYGGTTYYADSVKGRF DesignedHVR-H2sequence22: (SEQIDNO:74) VSGISGAGDTTYYADSVKGRF DesignedHVR-H2sequence23: (SEQIDNO:75) VSGISGDGDTTYYADSVKGRF DesignedHVR-H2sequence24: (SEQIDNO:76) VSGISGDGGSTYYADSVKGRF DesignedHVR-H2sequence25: (SEQIDNO:77) VSGISGYGDTTYYADSVKGRF DesignedHVR-H2sequence26: (SEQIDNO:78) VSGISGYGGTTYYADSVKGRF DesignedHVR-H2sequence27: (SEQIDNO:79) VSVISGDGDTTYYADSVKGRF DesignedHVR-H2sequence28: (SEQIDNO:80) VSVISGYGGSTYYADSVKGRF DesignedHVR-H2sequence29: (SEQIDNO:81) VSGISGDGSTTYYADSVKGRF DesignedHVR-H2sequence30: (SEQIDNO:82) VSGISGYGSTTYYADSVKGRF DesignedHVR-H2sequence31: (SEQIDNO:83) VSVISGSGSTTYYADSVKGRF DesignedHVR-H2sequence32: (SEQIDNO:84) VSVISGYGSSTYYADSVKGRF DesignedHVR-H2sequence33: (SEQIDNO:85) VSVISGYGSTTYYADSVKGRF DesignedHVR-H2sequence34: (SEQIDNO:86) VSAISGYGSTTYYADSVKGRF DesignedHVR-H2sequence35: (SEQIDNO:87) VSSISGYGDTTYYADSVKGRF DesignedHVR-H2sequence36: (SEQIDNO:88) VSSISGYGGSTYYADSVKGRF DesignedHVR-H2sequence37: (SEQIDNO:89) VSSISGYGGTTYYADSVKGRF DesignedHVR-H2sequence38: (SEQIDNO:90) VSYISGAGDTTYYADSVKGRF DesignedHVR-H2sequence39: (SEQIDNO:91) VSSISGAGDTTYYADSVKGRF DesignedHVR-H2sequence40: (SEQIDNO:92) VSYISGAGGTTYYADSVKGRF DesignedHVR-H2sequence41: (SEQIDNO:93) VSYISGDGDTTYYADSVKGRF DesignedHVR-H2sequence42: (SEQIDNO:94) VSYISGDGGSTYYADSVKGRF DesignedHVR-H2sequence43: (SEQIDNO:95) VSYISGDGGTTYYADSVKGRF DesignedHVR-H2sequence44: (SEQIDNO:96) VSYISGSGDTTYYADSVKGRF DesignedHVR-H2sequence45: (SEQIDNO:97) VSSISGAGGSTYYADSVKGRF DesignedHVR-H2sequence46: (SEQIDNO:98) VSYISGYGDTTYYADSVKGRF DesignedHVR-H2sequence47: (SEQIDNO:99) VSYISGYGGTTYYADSVKGRF DesignedHVR-H2sequence48: (SEQIDNO:100) VSSISGAGGTTYYADSVKGRF DesignedHVR-H2sequence49: (SEQIDNO:101) VSSISGDGDTTYYADSVKGRF DesignedHVR-H2sequence50: (SEQIDNO:102) VSSISGDGGTTYYADSVKGRF DesignedHVR-H2sequence51: (SEQIDNO:103) VSSISGAGSSTYYADSVKGRF DesignedHVR-H2sequence52: (SEQIDNO:104) VSSISGAGSTTYYADSVKGRF DesignedHVR-H2sequence53: (SEQIDNO:105) VSSISGDGSSTYYADSVKGRF DesignedHVR-H2sequence54: (SEQIDNO:106) VSSISGDGSTTYYADSVKGRF DesignedHVR-H2sequence55: (SEQIDNO:107) VSSISGYGSSTYYADSVKGRF DesignedHVR-H2sequence56: (SEQIDNO:108) VSSISGYGSTTYYADSVKGRF DesignedHVR-H2sequence57: (SEQIDNO:109) IGWINPNRGDTKYAQKFQGRV DesignedHVR-H2sequence58: (SEQIDNO:110) IGWINPNRGDTNYAQKFQGRV DesignedHVR-H2sequence59: (SEQIDNO:111) IGWINPNRGGTKYAQKFQGRV DesignedHVR-H2sequence60: (SEQIDNO:112) IGWINPNRGGTNYAQKFQGRV DesignedHVR-H2sequence61: (SEQIDNO:113) IGWINPNRGSTKYAQKFQGRV DesignedHVR-H2sequence62: (SEQIDNO:114) IGWINPNRGSTNYAQKFQGRV DesignedHVR-H2sequence63: (SEQIDNO:115) IGRINPNFGDTNYAQKFQGRV DesignedHVR-H2sequence64: (SEQIDNO:116) IGWINPNFGDTNYAQKFQGRV DesignedHVR-H2sequence65: (SEQIDNO:117) IGWINPNFGSTKYAQKFQGRV DesignedHVR-H2sequence66: (SEQIDNO:118) IGWINPNFGSTNYAQKFQGRV DesignedHVR-H2sequence67: (SEQIDNO:119) IGIINPNRGDTKYAQKFQGRV DesignedHVR-H2sequence68: (SEQIDNO:120) IGIINPNRGDTNYAQKFQGRV DesignedHVR-H2sequence69: (SEQIDNO:121) IGIINPNFGDTNYAQKFQGRV DesignedHVR-H2sequence70: (SEQIDNO:122) IGWISPSGGGTKYAQKFQGRV DesignedHVR-H2sequence71: (SEQIDNO:123) IGWISPSGGGTNYAQKFQGRV DesignedHVR-H2sequence72: (SEQIDNO:124) IGWISPSSGGTKYAQKFQGRV DesignedHVR-H2sequence73: (SEQIDNO:125) IGWISPSSGGTNYAQKFQGRV DesignedHVR-H2sequence74: (SEQIDNO:126) IGWIYPSGGGTKYAQKFQGRV DesignedHVR-H2sequence75: (SEQIDNO:127) IGWIYPSGGGTNYAQKFQGRV DesignedHVR-H2sequence76: (SEQIDNO:128) IGWISPSGGSTNYAQKFQGRV DesignedHVR-H2sequence77: (SEQIDNO:129) IGWISPSSGSTKYAQKFQGRV DesignedHVR-H2sequence78: (SEQIDNO:130) IGWISPSSGSTNYAQKFQGRV DesignedHVR-H2sequence79: (SEQIDNO:131) IGWISPSGGSTKYAQKFQGRV DesignedHVR-H2sequence80: (SEQIDNO:132) IGIIYPSGGGTNYAQKFQGRV DesignedHVR-H2sequence81: (SEQIDNO:133) IGIISPSGGGTKYAQKFQGRV DesignedHVR-H2sequence82: (SEQIDNo:134) IGIISPSGGGTNYAQKFQGRV DesignedHVR-H2sequence83: (SEQIDNO:135) IGIIYPSGGSTNYAQKFQGRV DesignedHVR-H2sequence84: (SEQIDNO:136) VGRIKSKTDGYTTEYAAPVKGRF DesignedHVR-H2sequence85: (SEQIDNO:159) VSAISGSGSTTYYADSVKGRF DesignedHVR-H2sequence86: (SEQIDNO:160) VSSISGSGDTTYYADSVKGRF DesignedHVR-H2sequence87: (SEQIDNO:161) VSSISGSGGSTYYADSVKGRF DesignedHVR-H2sequence88: (SEQIDNO:162) VSSISGSGGTTYYADSVKGRF DesignedHVR-H2sequence89: (SEQIDNO:163) VSSISGDGGSTYYADSVKGRF DesignedHVR-H2sequence90: (SEQIDNO:164) VSSISGSGSTTYYADSVKGRF FrameworkFW-H1sequence: (SEQIDNO:165) EVQLVESGGGLVQPGGSLRLSCAASG FrameworkFW-H2sequence: (SEQIDNO:166) RQAPGKGLEW FrameworkFW-H3sequence: (SEQIDNO:167) TISSRDNSKNTLYLQLNSLRAEDTAVYYC FrameworkFW-H4sequence: (SEQIDNO:168) WGQGTLVTVSS HitID4029VH (SEQIDNO:169) EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYHWGWIRQAPGKGLEWVSY ISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDYG DYYGFDYWGQGTLVTVSS HITID4029VL (SEQIDNO:170) DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLAWYQQKPGKAPKLL IYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTF GQGTKVEIKR HitID7097VH (SEQIDNO:171) EVQLVESGGGLVQPGGSLRLCAASGYSISSGHHWDWIRQAPGKGLEWVSYI SGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGSD AVLGDWFAYWGQGTLVTVSS HITID7097VL (SEQIDNO:172) DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGT KVEIKR HitID5906VH (SEQIDNO:173) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWNWIRQAPGKGLEWVSY ISGDGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLG GYYGWGRYFDYWGQGTLVTVSS HITID5906VL (SEQIDNO:174) DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIKR HitID7040VH (SEQIDNO:175) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWNWIRQAPGKGLEWIGW ISPSGGSTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLT AGGFDYWGQGTLVTVSS HITID7040VL (SEQIDNO:176) DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGT KVEIKR HitID5924VH (SEQIDNO:177) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWGWIRQAPGKGLEWIGI ISPSSGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGAG VHYALDYWGQGTLVTVSS HITID5924VL (SEQIDNO:178) DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIKR HitID4034VH (SEQIDNO:179) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNVVIRQAPGKGLEWVS SISGYGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARER YYGSTDYAFDYWGQGTLVTVSS HITID4034VL (SEQIDNO:180) DIQLTQSPSSLSASVGDRVTITCSASSRVSHVFWYQQKPGKAPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCLQGTHFPWTFGQGTK VEIKR HitID6010VH (SEQIDNO:181) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNVVIRQAPGKGLEWIG WINPNRGDTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDY YGDFDYWGQGTLVTVSS HITID6010VL (SEQIDNO:182) DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNVVYQQKPGKAPKLLIYD ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQG TKVEIKR HitID7183VH (SEQIDNO:183) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWVSS ISGYGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGS DTVLGDWFAYWGQGTLVTVSS HITID7183VL (SEQIDNO:184) DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNVVYQQKPGKAPKLLIYD ASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQG TKVEIKR HitID4036VH (SEQIDNO:185) EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGVGVGWIRQAPGKGLEWIG EIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARERY GSYYFDYWGQGTLVTVSS HITID4036VL (SEQIDNO:186) DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGKSFLDWYQQKPGKAPKLL IYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRIPPTF GQGTKVEIKR HitID5115VH (SEQIDNO:187) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWGWIRQAPGKGLEWIGE IYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARESYY AFDYWGQGTLVTVSS HITID5115VL (SEQIDNO:188) DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYAA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTPLTFGQGT KVEIKR HitID5404VH (SEQIDNO:189) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWAWIRQAPGKGLEWIGE IYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARSPYY YGVFDYWGQGTLVTVSS HITID5404VL (SEQIDNO:190) DIQLTQSPSSLSASVGDRVTITCSASSRVGSVYWYQQKPGKAPKLLIYDAS NLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYTHDPVTFGQGTK VEIKR HitID3757VH (SEQIDNO:191) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWI SPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYY GVGDFDYWGQGTLVTVSS HITID3757VL (SEQIDNO:192) DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIKR HitID5103VH (SEQIDNO:193) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWI SPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYY GVGDFDYWGQGTLVTVSS HITID5103VL (SEQIDNO:194) DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGT KVEIKR HitID4027VH (SEQIDNO:195) EVQLVESGGGLVQPGGSLRLSCAASGYSITSGHHWNVVIRQAPGKGLEWIG WISPSSGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGF DGFHYWGQGTLVTVSS HITID4027VL (SEQIDNO:196) DIQLTQSPSSLSASVGDRVTITCRASESVDFYGISFLPWYQQKPGKAPKLL IYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPWTF GQGTKVEIKR VHinFIG.1B (SEQIDNO:197) EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYGIHWVRQAPGKGLEWVSGI SGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARERDY DFDYWGQGTLVTVSS Formula(I) (SEQIDNO:198) X.sub.1TFX.sub.2X.sub.3YX.sub.4IHWV, whereinX.sub.1isForY,X.sub.2isSorT,X.sub.3isD,G,N, orS,andX.sub.4isA,G,orW Formula(II) (SEQIDNO:199) YSIX.sub.1SGX.sub.2X.sub.3WX.sub.4WI, whereinX.sub.1isSorT,X.sub.2isHorY,X.sub.3isHorY, andX.sub.4isA,D,G,N,S,orT Formula(III) (SEQIDNO:200) FSLSTX.sub.1GVX.sub.2VX.sub.3WI, whereinX.sub.1isGorS,X.sub.2isAorG,andX.sub.3isA,G, S,orT Formula(IV) (SEQIDNO:201) LAX.sub.1IX.sub.2WX.sub.3X.sub.4DKX.sub.5YSX.sub.6SLKSRL, whereinX.sub.1isLorR,X.sub.2isDorY,X.sub.3isA,D,S, orY,X.sub.4isDorG,X.sub.5isR,S,orY,andX.sub.6isP orT Formula(V) (SEQIDNO:202) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV, whereinX.sub.1isA,D,E,S,orY,X.sub.2isSorY,and X.sub.3isHorY Formula(VI) (SEQIDNO:203) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV, whereinX.sub.1isD,E,R,S,orY,X.sub.2isHorY,X.sub.3is NorS,andX.sub.4isNorY Formula(VII) (SEQIDNO:204) VSX.sub.1ISGX.sub.2GX.sub.3X.sub.4TYYADSVKGRF, whereinX.sub.1isA,G,S,V.orY,X.sub.2isA,D,S,or Y,X.sub.3isD,G,orS,andX.sub.4isSorT Formula(VIII) (SEQIDNO:205) IGX.sub.1INPNX.sub.2GX.sub.3TX.sub.4YAQKFQGRV, whereinX.sub.1isI,R,orW,X.sub.2isForR,X.sub.3isD,G, orS,andX.sub.4isKorN Formula(IX) (SEQIDNO:206) IGX.sub.1IX.sub.2PSX.sub.3GX.sub.4TX.sub.5YAQKFQGRV, whereinX.sub.1isI,R,orW,X.sub.2isSorY,X.sub.3isGor S,X.sub.4isD,G,orS,andX.sub.5isKorN Formula(X) (SEQIDNO:207) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTX.sub.5YAAX.sub.6VKGRF, whereinX.sub.1isKorR,X.sub.2isAorT,X.sub.3isDorY, X.sub.4isGorY,X.sub.5isDorE,andX.sub.6isPorS Formula(XI) (SEQIDNO:208) IGX.sub.1IX.sub.2X.sub.3SGSTYYSPSLKSRV, whereinX.sub.1isA,D,orE,X.sub.2isSorY,andX.sub.3isH orY Formula(XII) (SEQIDNO:209) IGX.sub.1IYX.sub.2SGX.sub.3TX.sub.4YNPSLKSRV, whereinX.sub.1isD,E,orS,X.sub.2isHorY,X.sub.3isNor S,andX.sub.4isNorY Formula(XIII) (SEQIDNO:210) VGRIX.sub.1SKX.sub.2X.sub.3GX.sub.4TTEYAAX.sub.5VKGRF, whereinX.sub.1isKorR,X.sub.2isAorT,X.sub.3isDorY, X.sub.4isGorY,X.sub.5isPorS PrimerF_1999 (SEQIDNO:211) CGTTTGTCCTGTGCAGCTTCCGG PrimerR_1999 (SEQIDNO:212) CGAGGCCCTTACCCGGGGCCTGACG PrimerF_2003 (SEQIDNO:213) CCGGGTAAGGGCCTCGAGTGG PrimerR_2003 (SEQIDNO:214) GAGCACGTCCGTTCGAATTGTCGCGACTTATAG PrimerS1089 (SEQIDNO:215) ACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTG PrimerS1090 (SEQIDNO:216) GAGGAGACGGTGACTAGTGTTCCTTGACCCCA PrimerF_2898 (SEQIDNO:217) TACTTATGTAGGCGATCGGGTCACCATCACCTGC PrimerR_2898 (SEQIDNO:218) CGGAGCTTTTCCTGGTTTCTGTTGATAC PrimerF_2013 (SEQIDNO:219) GAAACCAGGAAAAGCTCCGAAG PrimerR_2013 (SEQIDNO:220) CGTCCCGGAACCGGATCCAGAGAAGCGAG PrimerF2929 (SEQIDNO:221) ACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAAC PrimerR2929 (SEQIDNO:222) GATCTCCACCTTGGTACCCTGTCCGAA HVR-H3sequence1: (SEQIDNO:223) ARDLGGYYGWGRYFDY HVR-H3sequence2: (SEQIDNO:224) ARDLTAGGFDY HVR-H3sequence3: (SEQIDNO:225) ARDPGVGGFDV HVR-H3sequence4: (SEQIDNO:226) ARDPGYTWYFDV HVR-H3sequence5: (SEQIDNO:227) ARDYGDYYGFDY HVR-H3sequence6: (SEQIDNO:228) ARDYGYTWYFDV HVR-H3sequence7: (SEQIDNO:229) ARDYYGDFDY HVR-H3sequence8: (SEQIDNO:230) AREGSDAVLGDWFAY HVR-H3sequence9: (SEQIDNO:231) AREGSDTVLGDWFAY HVR-H3sequence10: (SEQIDNO:232) ARERYGSYYFDY HVR-H3sequence11: (SEQIDNO:233) ARERYYGSTDYAFDY HVR-H3sequence12: (SEQIDNO:234) ARESYYAFDY HVR-H3sequence13: (SEQIDNO:235) ARGAGVHYALDY HVR-H3sequence14: (SEQIDNO:236) ARGFDGFHY HVR-H3sequence15: (SEQIDNO:237) ARGFYGGALDV HVR-H3sequence16: (SEQIDNO:238) ARGGGGYYFDV HVR-H3sequence17: (SEQIDNO:239) ARGGGLGFDY HVR-H3sequence18: (SEQIDNO:240) ARGGLGPFDI HVR-H3sequence19: (SEQIDNO:241) ARGGSDTVIGDWFAY HVR-H3sequence20: (SEQIDNO:242) ARGGVGPFDI HVR-H3sequence21: (SEQIDNO:243) ARGGYGGYLDV HVR-H3sequence22: (SEQIDNO:244) ARGLSSGYFDY HVR-H3sequence23: (SEQIDNO:245) ARGSWYFDV HVR-H3sequence24: (SEQIDNO:246) ARGTRGLDY HVR-H3sequence25: (SEQIDNO:247) ARGYSDYFDY HVR-H3sequence26: (SEQIDNO:248) ARGYYYGRAFDY HVR-H3sequence27: (SEQIDNO:249) ARHSYYGVGDFDY HVR-H3sequence28: (SEQIDNO:250) ARLFEGFPY HVR-H3sequence29: (SEQIDNO:251) ARLYDYFAY HVR-H3sequence30: (SEQIDNO:252) ARSGYYALDY HVR-H3sequence31: (SEQIDNO:253) ARSPYYYGVFDY HVR-H3sequence32: (SEQIDNO:254) ARSYVYFDY HVR-H3sequence33: (SEQIDNO:255) ARDGLGLRGVYYYYYGLDV HVR-H3sequence34: (SEQIDNO:256) ARVGESGGIESPYYYYGLDV HVR-L1sequence1: (SEQIDNO:257) RASESVDFYGISFLP HVR-L1sequence2: (SEQIDNO:258) RASQSVDFYGISFLA HVR-L1sequence3: (SEQIDNO:259) RASQSVDFYGKSFLD HVR-L1sequence4: (SEQIDNO:260) SASSRVGSVY HVR-L1sequence5: (SEQIDNO:261) SASSRVSHVF HVR-L1sequence6: (SEQIDNO:262) RASQGISSYLA HVR-L1sequence7: (SEQIDNO:263) RASQSVSSYLA HVR-L1sequence8: (SEQIDNO:264) RASQSISSYLN HVR-L3sequence1: (SEQIDNO:265) FCLQGTHFPWT HVR-L3sequence2: (SEQIDNO:266) YCQQSYRTPFT HVR-L3sequence3: (SEQIDNO:267) YCQQSYSWPWT HVR-L3sequence4: (SEQIDNO:268) YCQQYTHDPVT HVR-L3sequence5: (SEQIDNO:269) YCQQYYRIPPT HVR-L3sequence6: (SEQIDNO:270) YCQHHYGTPLT HVR-L3sequence7: (SEQIDNO:271) YCQQSYSTPLT HVR-L3sequence8: (SEQIDNO:272) YCQQSYSTPPT HVR-L3sequence9: (SEQIDNO:273) YCQQYYSTPLT HVR-L3sequence10: (SEQIDNO:274) YCQQYYTTPLT