Dynamic human heavy chain antibody libraries

Abstract

To provide dynamic antibody libraries containing a robust set of dynamic units with well-defined developable sequence profiles for designing and constructing dynamic antibodies that are potentially more relevant functionally.SOLUTION: 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, where 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.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to libraries containing synthetic polynucleotides encoding antibody heavy chains (eg, heavy chains of dynamic human antibodies), as well as antibody heavy chains, antibodies, cells, animals, methods, and kits related thereto. [Background technology]

[0002] Monoclonal antibodies have become extremely useful in a variety of fields, including biological research, medical diagnostics, and medicine. The diversity of their potential binding specificities results in antibodies with useful specificities and capabilities. However, this diversity makes it difficult and tedious to screen the vast number of antibodies to identify one or more antibodies with the desired properties.

[0003] One method to identify antibodies of interest is to screen antibody libraries, such as libraries of cloned B cell sequences, phage display libraries, yeast display libraries, etc. These libraries allow screening of a large number of antibodies representing a large number of unique antibody sequences to identify antibodies with specific properties of interest, such as binding to a particular target, binding affinity, selectivity, etc. However, current libraries have certain limitations. Libraries derived from biological sources, such as human B cell repertoires, are limited to the antibody sequences that can be cloned from the biological source. Synthetic libraries, compared to biologically derived libraries, can contain non-natural sequences, but are also limited by the amount of antibodies that can be synthesized in a certain time frame. Furthermore, very large libraries require more time and exhaustive screening approaches, otherwise only a small portion of the library can actually be screened for the antibodies of interest.

[0004] Therefore, there is a need to develop dynamic antibody libraries that contain a sufficient set of dynamic units with well-defined exploitable sequence profiles to design and construct dynamic antibodies with potential functional relevance. Such libraries would greatly improve not only the diversity of antibody binding sites on the antibodies in the library, but also the efficiency of screening for antibodies with novel epitopes and / or conformational epitopes on a given antigen. Furthermore, such libraries would increase the likelihood of identifying specific antibodies of interest with high affinity and exploitability profiles.

[0005] All references cited in this specification, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] To meet these and other needs, disclosed herein are methods for determining the structure and function of heavy chain hypervariable regions (HVRs) and heavy chain variable regions (e.g., VVRs) that enable dynamic human antibodies. HThese antibody sequences are antibody sequences such as the HVR region. These sequences were designed to produce antibodies with highly flexible HVR sequence loops that can bind targets with high potency and / or recognize multiple useful epitopes and / or cross-react with epitopes shared between different species with low sequence identity (approximately 60% sequence identity or less). Advantageously, these antibody sequences allow the creation of smaller libraries that contain a large number of useful antibodies and / or have much greater diversity for a given library size. Such libraries can be used to identify novel antibodies of interest that are specific for a wide range of targets or, in some cases, cross-react with multiple targets of interest. Furthermore, a new concept and methodology for designing and constructing dynamic antibody libraries is introduced and implemented herein that uses the newly identified dynamic units to capture the wide conformational flexibility of antibody binding sites in a physically compact library. Furthermore, results using such antibodies (described below) highlight the ability to identify antibodies from these libraries that target conformational epitopes and / or evolutionarily conserved sites on a given antigen of different species with low sequence identity (e.g., less than 60%-70%).

[0007] Thus, in one aspect, provided herein are one or more HVR-H1 amino acid sequences and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding said amino acid sequences, wherein said HVR-H1 has a structure represented by formula (I): X1TFX2X3YX4IHWV (SEQ ID NO: 198), where X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W; and (III): FSLSTX1GVX2VX3WI (SEQ ID NO: 200) (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T). In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52.

[0008] In another aspect, provided herein are one or more HVR-H2 amino acid sequences and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding the amino acid sequences, wherein the HVR-H2 has the structure of formula (IV): LAX1IX2WX3X4DKX5YSX6SLKSRL (SEQ ID NO: 201), where 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); formula (V): IGX1IX2X3SGSTYYSPSLKSRV (SEQ ID NO: 202) (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y); formula (VI): IGX1IYX2SGX3TX4YNPSLKSRV (SEQ ID NO: 203) (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); formula (VII): VSX1ISGX2GX3X4TYYADS VKGRF (SEQ ID NO: 204) (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); formula (VIII): IGX1INPNX2GX3TX4YAQKFQGRV (SEQ ID NO: 205) (wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N); formula (IX): IGX1IX2PSX3GX4TX5YAQKFQGRV (SEQ ID NO: 206) (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); and (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (SEQ ID NO: 207) (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).In some embodiments, HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (IV); formula (VII); formula (VIII); formula (IX); formula (XI): IGX1IX2X3SGSTYYSPSLKSRV (SEQ ID NO: 208) (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y); formula (XII): IGX1IYX2SGX3TX4YNPSLKSRV (SEQ ID NO: 209) (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y); and formula (XIII): VGRIX1SKX2X3GX4TTEYAAX5VKGRF (SEQ ID NO: 210) (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). In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136.

[0009] In another aspect, provided herein are one or more HVR-H3 amino acid sequences, and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding the amino acid sequences, wherein the HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0010] In another aspect, provided herein are one or more HVR-L1 amino acid sequences, and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding the amino acid sequences, wherein the HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264.

[0011] In another aspect, provided herein are one or more HVR-L3 amino acid sequences, and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding the amino acid sequences, wherein the HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274.

[0012] In another aspect, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III). In some embodiments, provided herein are libraries comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of the polynucleotides in the library encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III).

[0013] In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, 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. In some embodiments, 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. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52.

[0014] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0015] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167.

[0016] In another aspect, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding an antibody heavy chain variable region comprising 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), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein are libraries comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X).

[0017] In some embodiments, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein are libraries comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the polynucleotides in the library encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII).

[0018] In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, 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, 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, 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.

[0019] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0020] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167.

[0021] In another aspect, provided herein is a polynucleotide (e.g., a synthetic polynucleotide) encoding an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III), and wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one of the polynucleotides in the library (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) encodes an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X).

[0022] In some embodiments, provided herein is a polynucleotide (e.g., a synthetic polynucleotide) encoding an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one of the polynucleotides in the library (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) encodes an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of the polynucleotides in the library encodes an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII).

[0023] In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. In some embodiments, 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, and 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, 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, and 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, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, and 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.

[0024] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VIII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (V); and HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XI); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XI); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VIII); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising an amino acid sequence of formula (II) and HVR-H2 comprising an amino acid sequence of formula (XIII); HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (XIII); and HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (XIII).

[0025] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 1 and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 122; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 154 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63. HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 145 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 128; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 153 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 67; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 100; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 123 R-H2; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 129; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 124; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 130;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 150 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 132; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 82; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 149 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of SEQ ID NO:26 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:151 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:34 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; HVR-H1 comprising the amino acid sequence of SEQ ID NO:5 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:6 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:116. HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:17 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; HVR-H1 comprising the amino acid sequence of SEQ ID NO:152 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 163; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 160; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:92; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:97; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 164; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 137 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 140 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 141 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 142 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 159; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 143 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 144 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 146 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 133; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 148 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118.

[0026] In some embodiments that may be combined with any of the preceding embodiments, the polynucleotides in the library are about 6.5*10 4 Less than (e.g., about 6.5*10 4 Less than 5.5*10 4 Less than 2.5*10 4 Less than 1*10 4In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) comprise no more than about 62272 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) comprise no more than about 60928 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) comprise no more than about 54656 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) comprise no more than about 6660 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) comprise no more than about 690 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, at least one of the HVR-H1 and HVR-H2 of the antibody heavy chain variable region adopts multiple conformations as assessed by structure determination and / or computer modeling.

[0027] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0028] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167. In some embodiments, 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.

[0029] In some embodiments, the polynucleotides in the library encode full-length antibody heavy chains. In some embodiments, the library further comprises one or more polynucleotides (e.g., synthetic polynucleotides) encoding an antibody light chain variable region. In some embodiments, the antibody light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the polynucleotides encoding the antibody light chain variable region comprise at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 1 ... 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 In some embodiments, one or more polynucleotides in the library that encode an antibody light chain variable region encodes a full-length antibody light chain.

[0030] In another aspect, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding a plurality of unique antibodies, each antibody comprising a heavy chain variable region and a light chain variable region, and wherein the heavy chain variable region of each of the plurality of antibodies comprises the same sequence and is encoded by any of the heavy chain variable region-encoding polynucleotides as described above. In some embodiments, provided herein are libraries comprising polynucleotides (e.g., synthetic polynucleotides) encoding a plurality of unique antibodies, each antibody comprising a heavy chain variable region and a light chain variable region, and wherein the heavy chain variable region of each of the plurality of antibodies comprises the same sequence and is encoded by any of the heavy chain variable region-encoding polynucleotides as described above.

[0031] In some embodiments, the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257 to 264, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265 to 274. In some embodiments, the light chain variable regions of the antibodies in the library comprise at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6 Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 Contains a unique sequence of

[0032] In another aspect, provided herein are vectors comprising any of the polynucleotides as described above. In some embodiments, provided herein are libraries comprising vectors, wherein at least one of the vectors in the library (e.g., at least one, at least two, at least five, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 690, at least 750, at least 1000, at least 2500, at least 5000, at least 6000, at least 6500, etc.) comprises any of the polynucleotides as described above. In some embodiments, at least two of the vectors in the library comprise a polynucleotide as described above. In some embodiments, at least 100 of the vectors in the library comprise a polynucleotide as described above. In some embodiments, at least 500 of the vectors in the library comprise a polynucleotide as described above. In some embodiments, at least 1000 of the vectors in the library comprise a polynucleotide as described above. In some embodiments, at least 5000 of the vectors in the library comprise a polynucleotide as described above. In some embodiments, at least 6500 of the vectors in the library comprise a polynucleotide as described above. In some embodiments, provided herein is a library comprising vectors, each of the vectors in the library comprising any of the polynucleotides described above. In some embodiments, the vectors are expression vectors. In some embodiments, the vectors are display vectors. In some embodiments, the library comprising vectors further comprises at least one (e.g., at least 1, at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 690, at least 750, at least 1000, at least 2500, at least 5000, at least 6000, at least 6500, etc.) vector encoding a light chain variable region polypeptide.In some embodiments, the light chain variable region comprises HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257 to 264 and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 265 to 274. In some embodiments, at least one vector in the library contains at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10. 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6 Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 The light chain variable region contains a unique sequence:

[0033] In another aspect, provided herein are cells comprising any of the polynucleotides and / or vectors as described above. In some embodiments, provided herein are libraries comprising a population of cells, wherein at least one (e.g., at least 1, at least 2, at least 5, at least 10, at least 100, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9etc.) comprise any of the polynucleotides and / or vectors as described above. In some embodiments, at least two of the cells in the library comprise a polynucleotide and / or vector as described above. In some embodiments, at least 100 of the cells in the library comprise a polynucleotide and / or vector as described above. In some embodiments, provided herein are libraries comprising a population of cells, each of the cells in the library comprises any of the polynucleotides and / or vectors as described above. In some embodiments, the cells are bacterial, yeast, or mammalian cells (e.g., non-human animal cells or isolated human cells).

[0034] In another aspect, provided herein is an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the heavy chain variable regions in the library comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of the heavy chain variable regions in the library comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III).

[0035] In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, 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. In some embodiments, 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. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52.

[0036] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0037] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167.

[0038] In another aspect, provided herein is an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the heavy chain variable regions in the library comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of the heavy chain variable regions in the library comprises 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), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X).

[0039] In some embodiments, provided herein are antibody heavy chain variable regions comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein are libraries comprising antibody heavy chain variable regions, wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the heavy chain variable regions in the library comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of the heavy chain variable regions in the library comprises 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), formula (VII), formula (VIII), formula (IX), formula (XI), formula (XII), and formula (XIII).

[0040] In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, 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, 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, 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.

[0041] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0042] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167.

[0043] In another aspect, provided herein is an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the heavy chain variable regions in the library comprises 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of the heavy chain variable regions in the library comprises 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X).

[0044] In some embodiments, provided herein is an antibody heavy chain variable region comprising 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, wherein at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) of the heavy chain variable regions in the library comprises 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of the heavy chain variable regions in the library comprises 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), Formula (II), and Formula (III), and the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of Formula (IV), Formula (VII), Formula (VIII), Formula (IX), Formula (XI), Formula (XII), and Formula (XIII).

[0045] In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. In some embodiments, 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, and 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, 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, and 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, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, and 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.

[0046] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VIII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (V); and HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XI); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XI); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VIII); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising an amino acid sequence of formula (II) and HVR-H2 comprising an amino acid sequence of formula (XIII); HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (XIII); and HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (XIII).

[0047] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 1 and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 122; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 154 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63. HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 145 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 128; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 153 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 67; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 100; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 123 R-H2; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 129; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 124; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 130;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 150 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 132; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 82; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 149 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of SEQ ID NO:26 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:151 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:34 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; HVR-H1 comprising the amino acid sequence of SEQ ID NO:5 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:6 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:116. HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:17 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; HVR-H1 comprising the amino acid sequence of SEQ ID NO:152 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 163; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 160; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:92; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:97; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 164; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 137 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 140 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 141 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 142 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 159; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 143 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 144 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 146 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 133; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 148 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118.

[0048] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0049] In some embodiments, the heavy chain variable region further comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 comprising the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 comprising the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises at least two (e.g., at least two, at least three, or all four) of FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and FW-H4 comprising the amino acid sequence of SEQ ID NO: 168, in any combination. In some embodiments, the FW-H3 sequence comprises an arginine to lysine mutation at R19 of SEQ ID NO: 167. In some embodiments, 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.

[0050] In some embodiments that may be combined with any of the preceding embodiments, the heavy chain variable regions in the library are about 6.5*10 4 Less than (e.g., about 6.5*10 4 Less than 5.5*10 4 Less than approx. 2.5*10 4 Less than 1*10 4less 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. In some embodiments, the heavy chain variable regions in the library comprise no more than about 62272 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the heavy chain variable regions in the library comprise no more than about 60928 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the heavy chain variable regions in the library comprise no more than about 54656 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the heavy chain variable regions in the library comprise no more than about 6660 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the heavy chain variable regions in the library comprise no more than about 690 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, at least one of the HVR-H1 and HVR-H2 of the antibody heavy chain variable regions adopts multiple conformations as assessed by structure determination and / or computer modeling.

[0051] In another aspect, provided herein are antibody heavy chain variable regions and antibody light chain variable regions, wherein the antibody heavy chain variable region is any of the heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antibody heavy chain variable regions and antibody light chain variable regions, wherein at least one (e.g., at least one, at least two, at least five, at least ten, at least one hundred, etc.) of the antibody heavy chain variable regions in the library is any of the heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antibody heavy chain variable regions and antibody light chain variable regions, wherein each of the antibody heavy chain variable regions in the library is any of the heavy chain variable regions as described herein. In some embodiments, the antibody light chain variable region comprises HVR-L1, HVR-L2, and 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. In some embodiments, the light chain variable regions in the library contain at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6 Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 Contains a unique sequence of

[0052] In another aspect, provided herein are antigen-binding domains comprising an antibody heavy chain variable region, the antigen-binding domains comprising any of the antibody heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antigen-binding domains comprising an antibody heavy chain variable region, at least one (e.g., at least one, at least two, at least five, at least ten, at least one hundred, etc.) of the antigen-binding domains in the library comprises any of the heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antigen-binding domains comprising an antibody heavy chain variable region, each of the antigen-binding domains in the library comprises any of the heavy chain variable regions as described herein. In some embodiments, the antigen-binding domains further comprise an antibody light chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, the HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and / or the HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the antigen binding domains comprising light chain variable regions in the library comprise at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6 Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 The light chain variable region comprises a unique sequence of:

[0053] In another aspect, provided herein are antibodies comprising an antibody heavy chain variable region, the antibody comprising any of the antibody heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antibodies, at least one (e.g., at least one, at least two, at least five, at least ten, at least one hundred, etc.) of the antibodies in the library comprises any of the antibody heavy chain variable regions as described herein. In some embodiments, provided herein are libraries comprising antibodies, each of the antibodies in the library comprises any of the heavy chain variable regions as described herein. In some embodiments, the antibody further comprises an antibody light chain variable region comprising HVR-L1, HVR-L2, and HVR-L3, the HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and / or the HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the antibodies comprising the light chain variable regions in the library have at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10 3 Unique sequences of at least 10 4 Unique sequences of at least 10 5 Unique sequences of at least 10 6 Unique sequences of at least 10 7 Unique sequences of at least 10 8 or at least about 10 unique sequences 9 The light chain variable region comprises a unique sequence of:

[0054] In some embodiments that may be combined with any of the preceding embodiments, the antibody has a mAb concentration of about 6.5*10 4 In some embodiments that may be combined with any of the preceding embodiments, the antibody comprises a unique combination of HVR-H1 and HVR-H2 sequences that is less than about 5.5*10 4In some embodiments, the antibody comprises no more than about 62272 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the antibody comprises no more than about 60928 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the antibody comprises no more than about 54656 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the antibody comprises no more than about 6660 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, the antibody comprises no more than about 690 unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, at least one of the HVR-H1 and HVR-H2 of the antibody heavy chain variable region adopts multiple conformations as assessed by structure determination and / or computer modeling.

[0055] In some embodiments that may be combined with any of the preceding embodiments, the antibody is -7 ~about 10 -11 It binds to at least one target with an equilibrium dissociation constant (Kd) of M. In some embodiments, the antibody has a melting temperature (Tm) of about 60°C to about 90°C.

[0056] In another aspect, provided herein are polypeptides (e.g., scaffold polypeptides) that comprise one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) HVRs of the disclosure. In some embodiments, provided herein are libraries comprising polypeptides, where at least one of the polypeptides in the library (e.g., at least one, at least two, at least five, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 2500, at least 5000, at least 6000, at least 6500, etc.) comprises one or more HVRs of the disclosure. In some embodiments, provided herein are libraries comprising polypeptides, where each of the polypeptides in the library comprises one or more HVRs of the disclosure. In some embodiments, the polypeptide comprises an HVR-H1 comprising an amino acid sequence selected from any HVR-H1 sequence as described herein (e.g., an HVR-H1 according to a formula selected from the group consisting of Formula (I), Formula (II), and Formula (III); 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 as described herein (e.g., an HVR-H2 according to a formula 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); 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 as described herein (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 as described herein (e.g., SEQ ID NOs: 257-264).In some embodiments, a polypeptide comprises an HVR-L3 comprising an amino acid sequence selected from any of the HVR-L3 sequences as described herein (e.g., SEQ ID NOs: 265-274). In some embodiments, a 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, provided herein are polynucleotides encoding any of such polypeptides, and libraries comprising polynucleotides.

[0057] In another aspect, provided herein is a phage comprising at least one polypeptide on its surface, the at least one polypeptide comprising any of the antibody heavy chain variable regions described herein. In some embodiments, the at least one polypeptide is any of the antigen binding domains as described herein. In some embodiments, provided herein is a library of phage, wherein at least one of the phage in the library (e.g., at least 1, at least 2, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 2500, at least 5000, at least 6000, at least 6500, etc.) comprises at least one polypeptide comprising any of the antibody heavy chain variable regions described herein on its surface. In some embodiments, at least one phage in the library comprises at least one polypeptide comprising any of the antigen binding domains as described herein on its surface. In some embodiments, provided herein is a library comprising phage, each of the phage in the library comprising on its surface at least one polypeptide comprising any of the antibody heavy chain variable regions described herein, in some embodiments, the at least one polypeptide is any of the antigen binding domains as described herein.

[0058] In another aspect, provided herein is a non-human animal comprising at least one polynucleotide (e.g., any of the polynucleotides or polynucleotide libraries described herein) encoding any of the antibody heavy chain variable regions described herein. In some embodiments, the non-human animal comprises at least one polynucleotide encoding any of the antibodies described herein. In some embodiments, the non-human animal is a mammal (e.g., a mouse, a rat, a rabbit, a camel, or a non-human primate).

[0059] In another aspect, provided herein is a method of preparing a library comprising providing and assembling any of the polynucleotide sequences of the library as described herein.

[0060] In another aspect, provided herein is a method of screening for polypeptides that bind to a target, comprising incubating any of the libraries comprising the polypeptides described herein (e.g., a library of antigen binding domains, a library of antibodies, a library of phage, etc.) with the target, and selecting one or more polypeptides from the library that bind to the target.

[0061] In another aspect, provided herein is a method for generating an antibody library, comprising: (a) selecting one, two or three heavy chain HVRs that contain sequences with multiple conformations; and (b) assembling polynucleotide sequences to generate a library of synthetic polynucleotides encoding multiple antibody heavy chain variable region sequences. In some embodiments, at least one of the multiple antibody heavy chain variable region sequences is any of the heavy chain variable region sequences described herein. In some embodiments, each of the multiple antibody heavy chain variable region sequences is any of the heavy chain variable region sequences described herein.

[0062] In another aspect, provided herein is a method for preparing a polypeptide (e.g., a heavy chain variable region, an antibody heavy chain, an antibody, a scaffold polypeptide, etc.) comprising culturing a cell comprising any of the polynucleotides, polynucleotide libraries, vectors, and / or vector libraries as described above to produce the polypeptide. In some embodiments, the polypeptide is recovered from the cultured cells and further purified.

[0063] In another aspect, provided herein is a method for making 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, the first antigen-binding domain comprising a first antibody heavy chain variable region and a first antibody light chain variable region, the first antibody heavy chain variable region comprising any of the heavy chain variable regions described herein; (b) screening for a second antigen-binding domain that binds to a second antigen, the second antibody heavy chain variable region and a second antibody light chain variable region, the second antibody heavy chain variable region having the same sequence as the first antibody heavy chain variable region; and (c) generating a bispecific antibody comprising the first antigen-binding domain and the second antigen-binding domain.

[0064] In another aspect, provided herein is a bispecific antibody comprising: (a) a first binding domain comprising a first heavy chain variable region and a first light chain variable region, the first binding domain binding to a first target; and (b) a second binding domain comprising a second heavy chain variable region and a second light chain variable region, the second binding domain binding to a second target, the second heavy chain variable region having a sequence identical to the first heavy chain variable region sequence, each of the first and second heavy chain variable regions comprising any of the heavy chain variable regions described herein. In some embodiments, the bispecific antibody comprises a first light chain and a second light chain, the first light chain comprising a first light chain variable region and the second light chain comprising a second light chain variable region, each of the first and second light chains comprising a kappa C L domain (e.g., human kappa C L In some embodiments, the bispecific antibody comprises a first light chain and a second light chain, the first light chain comprising a first light chain variable region and the second light chain comprising a second light chain variable region, each of the first and second light chains comprising a lambda C domain. L domain (e.g., human lambda C LIn some embodiments, the bispecific antibody comprises a first light chain and a second light chain, the first light chain comprising a first light chain variable region and a kappa C domain. L domain (e.g., human kappa C L domain), the second light chain comprising a second light chain variable region and a lambda C L domain (e.g., human lambda C L In some embodiments, the bispecific antibody comprises a first light chain and a second light chain, the first light chain comprising a first light chain variable region and a lambda C domain. L domain (e.g., human lambda C L domain), the second light chain comprising a second light chain variable region and a kappa C L domain (e.g., human kappa C L domain).

[0065] In another aspect, provided herein are kits comprising any of the polynucleotides, polynucleotide libraries, vectors, and / or vector libraries (or any cell or population of cells containing them) as described herein. In some embodiments, provided herein are kits comprising any of the heavy chain variable regions, heavy chain variable region libraries, antigen binding domains, antigen binding domain libraries, antibodies, antibody libraries, polypeptides (e.g., scaffold polypeptides), polypeptide libraries, phages, and / or phage libraries as described herein.

[0066] It should be understood that one, more than one, or all of the characteristics of the various embodiments described above and herein may be combined to form other embodiments of the present invention. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further detailed in the following detailed description. [Brief description of the drawings]

[0067] [Figure 1]Figure 1A shows an entropy plot of the amino acids of the VH domain by residue number. The VH structures of 113 human antibodies were used to calculate the entropy. Figure 1B shows the definition of hypervariable region (HVR) used herein for an exemplary antibody heavy chain variable domain (VH) sequence (SEQ ID NO: 197) compared to the Kabat definition of the complementarity determining region (CDR) of the same VH sequence. [Figure 2A] The affinity measurements of fabs confirmed to bind to the antigens TAGT-1 to TAGT-12 are shown. [Figure 2B] The measured melting temperatures (Tm) of fabs confirmed to bind to the antigens TAGT-1 to TAGT-12 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present disclosure provides libraries containing synthetic (e.g., non-natural) polynucleotides encoding antibody heavy chains (e.g., heavy chains of dynamic human antibodies). Advantageously, the antibody heavy chains disclosed herein contain HVR sequences designed to generate flexible loops for more effective substrate binding and / or specificity for multiple substrates of interest. These HVR sequences allow for the creation of smaller antibody libraries with broader epitope coverage than existing techniques.

[0069] I. General techniques The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and may be implemented using conventional methodology, e.g., as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987);PCR: The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(JEColigan et al.,eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (CA 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 JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., J.B. Lippincott Company, 1993).

[0070] II. Definition Before describing the present disclosure in detail, it is to be understood that this 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 used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0071] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and so forth.

[0072] The term "about" as used herein refers to the normal error range for the respective value, which is readily understood by one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to the value or parameter itself.

[0073] It is to be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0074] The term "antibody" is used herein in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., single-chain variable fragments or scFvs), so long as they exhibit the desired biological activity.

[0075] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. H and V L and form a single antigen-binding site. For the structure and properties of different antibody classes, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0076] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain (CH) of their heavy chains. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"). The gamma and alpha classes are further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different immunoglobulin classes are known; see, e.g., Abbas et al., Cellular and Molecular Immunology, 4 th This is outlined in Saunders Co., ed. (WB Saunders Co., 2000).

[0077] 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 is referred to as "V H The light chain variable domain is sometimes called the "V L These domains are generally the most variable parts of antibodies and contain the antigen-binding sites.

[0078] The term "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used in Kabat et al. (supra) to organize antibody heavy or light chain variable domains. When using this numbering system, the actual linear amino acid sequence may contain fewer amino acids depending on the truncation of the FR or HVR of the variable domain, or may contain additional amino acids depending on the insertion into the FR or HVR. For example, a heavy chain variable domain may contain one amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may contain multiple inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of residues for a given antibody may be determined by alignment of the antibody's sequence with the "standard" Kabat numbering sequence at the regions of homology.

[0079] The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in 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 commonly used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index according to Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0080] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain is the C H 1. C H 2 and C HIt contains 3 domains (collectively, CH) and a light chain CHL (or CL) domain.

[0081] The term "full length antibody" (the terms "intact" or "whole" antibody may be used interchangeably herein) may refer to an antibody in substantially intact form, as opposed to an antibody fragment. Similarly, the term "full length antibody heavy chain" (the terms "intact" or "whole" antibody heavy chain may be used interchangeably herein) may refer to an antibody heavy chain in substantially intact form, as opposed to an antibody heavy chain fragment. In particular, whole antibodies include those having 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, an intact antibody may have one or more effector functions.

[0082] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogenous population of antibodies. That is, the individual antibodies in the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include a variety of 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 homogenous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure can be made by a variety of techniques, including, for example, the hybridoma method (see, 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, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display methods (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 352:624-628 (1991)), and the like. 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 techniques for producing human antibodies or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (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. Patent 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).

[0083] As used herein, "hypervariable region (HVR)" refers to a region of an antibody domain that is sequence hypervariable and / or forms a structurally defined loop. Generally, an antibody contains 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, NJ, 2003). Each VH and VL is composed of three HVRs and four framework (FW) regions, arranged from the amino terminus to the carboxy terminus in the following order: FW1-HVR1-FW2-HVR2-FW3-HVR3-FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Throughout this 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 HVRs (as used herein) of the exemplary antibody heavy chain variable domain shown in FIG. 1B differs from the Kabat definition of 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).

[0084] As used herein, a "library" refers to a collection of two or more entities that have a common class. For example, a library containing polynucleotides can refer to a collection of two or more polynucleotides. The term "library" is used herein in its broadest sense and specifically includes sub-libraries that may or may not be combined.

[0085] As used herein, "unique" refers to a member of a collection that is different from other members of the collection. For example, a unique antibody derived from a library encoding a plurality of polynucleotides encoding antibodies may refer to an antibody that has a specific sequence that is not shared by other antibodies encoded by the library. It should be understood that, as a practical matter, a "unique" member physically realized by a library may exist in more than one copy. For example, a library may contain multiple "unique" antibodies, and one or more of the "unique" antibody molecules may occur in more than one copy.

[0086] As used herein, "diversity" refers to variability and / or heterogeneity. For example, the diversity of antibodies in a library can refer to the variety of antibodies that contain unique sequences present in the library.

[0087] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and may refer to a polymer of two or more amino acids.

[0088] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including 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 can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can contain 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 natural nucleotides with analogs, internucleotide modifications, such as uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications containing intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylators, modifications with modified bonds (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, activated to generate further bonds to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, substituted or unsubstituted alkyl (1-20C) (optionally containing ether (-O-) linkages), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages within a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0089] A cell (e.g., a cell or population of cells containing a synthetic polynucleotide or a library of synthetic polynucleotides) includes an individual cell or cell culture that can be or has been the recipient of a vector(s) for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. A host cell includes cells transfected in vivo with a polynucleotide(s) (e.g., a synthetic polynucleotide encoding an antibody heavy chain variable region of the present disclosure).

[0090] "Non-human animal" refers to any animal not classified as a human, such as livestock, farm or zoo animals, sport animals, pet animals (dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to, but are not limited 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 a preferred embodiment, the animal is an antibody-producing animal.

[0091] III. Antibody Libraries and Library Generation Certain aspects of the present disclosure relate to polynucleotides, such as antibody heavy chain variable regions (V H ) or the light chain variable region (V L The disclosed libraries may contain one or more polynucleotides encoding heavy chain variable regions comprising HVR-H1, HVR-H2, and HVR-H3, where HVR-H1 and HVR-H2 are any of the HVR-H1 and / or HVR-H2 described herein.

[0092] In some embodiments, the libraries of the present disclosure contain a smaller number of unique heavy chain HVR sequences and / or unique VVR sequences than a typical antibody library. H Advantageously, such libraries can provide sufficient diversity to identify antibodies that bind to one or more of a large number of antigens of interest, while allowing for efficient screening due to the reduced library size. In some embodiments, the libraries of the disclosure comprise or consist of polynucleotides that comprise less than about 10,000, less than about 9,000, less than about 8,000, or less than about 7,000 unique combinations of HVR-H1 and HVR-H2 sequences. In certain embodiments, the libraries of the disclosure comprise or consist of polynucleotides that comprise no more than about 6,600 unique combinations of HVR-H1 and HVR-H2 sequences.

[0093] In some embodiments, a library contains a plurality of polynucleotides, at least one of which encodes an antibody heavy chain variable region of the disclosure (e.g., comprising HVR-H1 and HVR-H2 of the disclosure).

[0094] In some embodiments, one or more of the polynucleotides encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is selected from the group consisting of: (Formula I) X1TFX2X3YX4IHWV, where 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) YSIX1SGX 2X3WX4WI, where 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, where 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 encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 is selected from the group consisting of: (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where 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) IGX 1IYX2SGX3TX4YNPSLKSRV, where 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, where 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, where 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, where 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, where 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, HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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 encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is of formula I: X1TFX2X3YX4IHWV, where 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); or of formula II: YSIX1SGX2X3WX4WI, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, and X3 is A, D, S, or Y. and X4 is D or G, X5 is R, S, or Y, and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where X1 is D, E, R, S, or Y wherein 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) IGX1INPNX2GX3TX4YAQ KFQGRV, where 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, where 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, where 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 encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is selected from the group consisting of: (Formula I) X1TFX2X3YX4IHWV, where 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, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200). HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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 a display vector);

[0095] 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 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, including HVR-H1, HVR-H2, and HVR-H3. and the HVR-H1 encodes an antibody heavy chain variable region comprising: (Formula I) X1TFX2X3YX4IHWV, where 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, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and / or (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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). No. 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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, where X1 is A, 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, where X1 is I, R, or W, X2 is S or Y, and X4 is S or T (SEQ ID NO: 204); (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV, where 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, where X1 is I, R, or W, X2 is S or Y, and 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 about 6.5*10 of the polynucleotides; 4 Less than (e.g., about 6.5*10 4 Less than 5.5*10 4 Less than 2.5*10 4Less than 1*10 4less 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.), 62272 or less, 60928 or less, 54656 or less, or 6660 or less encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is represented by the sequence of (formula I) X1TFX2X3YX4IHWV, where X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W (sequence (Formula II) YSIX1SGX2X3WX4WI, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200), and (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X 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, where 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, where X1 is D, E, R, S, or Y, X2 is H or Y, and X3 is H or Y) (SEQ ID NO: 203). is N or S, and X4 is N or Y) (SEQ ID NO: 203); (Formula VII) VSX1ISGX2GX3X4TYYADSVKGRF, where 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, where 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);HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV, where 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, where 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 a display vector);

[0096] 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 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, including HVR-H1, HVR-H2, and HVR-H3. and the HVR-H1 encodes an antibody heavy chain variable region comprising: (Formula I) X1TFX2X3YX4IHWV, where 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, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and / or (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, where X1 is 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, where 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 about 6.5*10 of the polynucleotides; 4 Less than (e.g., about 6.5*10 4 Less than 5.5*10 4 Less than 2.5*10 4 Less than 1*10 4less 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.), 62272 or less, 60928 or less, 54656 or less, or 6660 or less encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is represented by the formula I: X1TFX2X3YX4IHWV, where X1 is F or Y. and 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, where 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, where X1 is G or S, and X (X1 is A or G, and X2 is S or Y, and X3 is H or Y) (SEQ ID NO:205); (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO:206); (Formula XI) IGX1IYX2SGX3TX4YNPSLKSRV, where X1 is D, E, or S and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF, where 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 a display vector).

[0097] In some embodiments, the polynucleotides in the library comprise a HVR-H1 comprising an amino acid sequence according to the formula X1TFX2X3YX4IHWV, where 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 a HVR-H2 comprising an amino acid sequence according to (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, and X5 is R, S, or W. or Y, and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) VSX1ISGX2GX3X4TYY ADSVKGRF, where 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, where 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, where X1 is I, R, or W, 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).

[0098] In some embodiments, the polynucleotides in the library comprise an HVR-H1 comprising an amino acid sequence according to the formula X1TFX2X3YX4IHWV, where 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 XI)IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3T and HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: X4YNPSLKSRV, where 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, where 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).

[0099] In some embodiments, the polynucleotides in the library comprise a HVR-H1 comprising an amino acid sequence according to the formula YSIX1SGX2X3WX4WI, where 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 a HVR-H2 comprising an amino acid sequence according to (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, and X5 is R, S, or T. or Y, and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) VSX1ISGX2GX3X4TYY ADSVKGRF, where 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, where 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, where X1 is I, R, or W, 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).

[0100] In some embodiments, the polynucleotides in the library comprise an HVR-H1 comprising an amino acid sequence according to the formula YSIX1SGX2X3WX4WI, where 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); (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3 and HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: TX4YNPSLKSRV, where 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, where 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).

[0101] In some embodiments, the polynucleotides in the library comprise a HVR-H1 comprising an amino acid sequence according to the formula FSLSTX1GVX2VX3WI, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and a HVR-H2 comprising an amino acid sequence according to the formula FSLSTX1GVX2VX3WI, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200). or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) VSX1ISGX2GX3X4TYYADSVKGR F, where 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, where 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, where X1 is I, R, or W, X2 is F or R, X1 is S or Y, X2 is G or S, X4 is D, G, or S, and X5 is K or N (SEQ ID NO: 206); and HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, where Xi 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).

[0102] In some embodiments, the polynucleotides in the library comprise an HVR-H1 comprising an amino acid sequence according to the formula FSLSTX1GVX2VX3WI, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XI) IGX1IYX2SGX3TX4YNPSL and HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: KSRV, where 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, where 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).

[0103] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising 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 to 52 and 137 to 158. In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising 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 to 52.

[0104] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising 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 to 136 and 159 to 164. In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising 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 to 136.

[0105] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of Formula (I), Formula (II), and Formula (III), or wherein 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, the polynucleotide library encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, or wherein HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, 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 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, 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 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, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, or 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.

[0106] In some embodiments, the polynucleotide library encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, where HVR-H3 is any HVR-H3 known in the art. In some embodiments, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.

[0107] The heavy chain HVR sequences described herein may be included in any combination in the library of the present disclosure. In some embodiments, the heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, the heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, the heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, the heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.In some embodiments, the heavy chain variable region comprises HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.

[0108] In certain embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, and the HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (II) HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VIII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (V);In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XI); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XI); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VIII); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising an amino acid sequence of formula (II) and HVR-H2 comprising an amino acid sequence of formula (XIII); HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (XIII); and HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (XIII).

[0109] In certain embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 1 and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 122; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 154 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 145 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 128; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 153 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 67; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 100; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 123 VR-H2; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 129; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 124; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 130;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 150 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 132; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 82; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 149 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of SEQ ID NO:26 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:151 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:34 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; HVR-H1 comprising the amino acid sequence of SEQ ID NO:5 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:6 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:116. HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:17 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; HVR-H1 comprising the amino acid sequence of SEQ ID NO:152 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 163; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 160; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:92; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:97; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 164; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 137 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 140 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 141 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 142 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 159; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 143 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 144 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 146 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 133; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 148 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118.

[0110] In some embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, where HVR-H1 and HVR-H2 are listed in Table 1. In some embodiments, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, 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, 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.

[0111] In some embodiments, the heavy chain variable region further comprises variable region heavy chain framework sequences located adjacent to and between the HVRs according to the following 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 are: FW-H1 is EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 165), FW-H2 is RQAPGKGLEW (SEQ ID NO: 166); FW-H3 is TISRDSNSKNTLYLQLNSLRAEDTAVYYC (SEQ ID NO: 167); FW-H4 is WGQGTLVTVSS (sequence number 168).

[0112] In some embodiments, the heavy chain variable region comprises an alternative 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 are FW-H1 of SEQ ID NO: 165, FW-H2 of SEQ ID NO: 166, SEQ ID NO: 167 or FW-H3 with an arginine to lysine mutation at R19, and FW-H4 of SEQ ID NO: 168.

[0113] In some embodiments, the library contains a plurality of polynucleotides, at least one of which encodes an antibody light chain variable region (e.g., comprising HVR-L1, HVR-L2, and HVR-L3). In some embodiments, the antibody light chain variable region comprises 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 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 HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264 and HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 265-274. In some embodiments, the library contains at least 1, at least 50, at least 100, at least 250, at least 500, at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 In some embodiments, the library contains a plurality of polynucleotides encoding unique sequences of antibody light chain variable regions of at least 10 3 In some embodiments, the library contains a plurality of polynucleotides encoding unique sequences of antibody light chain variable regions of at least 10 5 In some embodiments, the library contains a plurality of polynucleotides encoding unique sequences of antibody light chain variable regions of at least 10 9 In another embodiment, the library contains a polynucleotide encoding one antibody light chain variable region. In some embodiments, the library contains 1 to about 10 polynucleotides encoding the unique sequences of the antibody light chain variable regions. 3The antibody light chain variable region comprises a plurality of polynucleotides encoding unique sequences of an antibody light chain variable region of the present invention. In some embodiments, the antibody light chain variable region is any of the antibody light chain variable regions found in patent applications filed with Attorney Docket Nos. 69540-3000100, 69540-2000140, and / or 69540-2000100, the disclosures of which are each incorporated 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 patent applications filed with Attorney Docket Nos. 69540-3000100, 69540-2000140, and / or 69540-2000100, the disclosures of which are each incorporated by reference in their entireties.

[0114] In some embodiments, one or more of the polynucleotides in the library encode a full-length antibody heavy chain(s). In other embodiments, one or more of the polynucleotides in the library encode a heavy chain Fab fragment(s). In some embodiments, one or more of the polynucleotides in the library encode a single chain variable fragment(s).

[0115] In some embodiments, the library contains a plurality of polynucleotides encoding 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 of the plurality of antibodies comprises an identical sequence, comprising HVR-H1, HVR-H2 and HVR-H3. In some embodiments, at least one or at least two of HVR-H1 and HVR-H2 are HVR-H1 sequences of the disclosure (e.g., X1TFX2X3YX4IHWV, where 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, where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, G, or W (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 HVR-H2 sequences of the disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, and X3 is A, D, 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 HVR-H2 sequences of the disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, and X3 is A, D, S, or T) (SEQ ID NO: X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:202); IGX1IYX2SGX3TX4YNPSLKSRV, where X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or T (SEQ ID NO:203); IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO:204); is 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, where 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, where 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, where X1 is A, D, or E, and X2 is K or N (SEQ ID NO: 208); X1 is S or Y, and X3 is H or Y (SEQ ID NO: 208); IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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 amino acid sequences selected from 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 disclosure that also includes a polynucleotide encoding one or more light chain variable region(s).

[0116] In some embodiments, a library of the disclosure comprises one or more vectors encoding one or more polynucleotides (eg, synthetic polynucleotides) of the disclosure.

[0117] Further provided herein are methods of preparing a library, e.g., by providing and assembling polynucleotide sequences (e.g., synthetic polynucleotide(s)) of the library of the disclosure. Further provided herein are methods of making a library, e.g., including selecting one, two, or three heavy chain HVRs (e.g., one or two heavy chain HVRs of the disclosure) that include sequences with multiple conformations, and assembling the polynucleotide sequences to generate a library of polynucleotides (e.g., synthetic polynucleotides) encoding multiple 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 HVR-H1, HVR-H2 and HVR-H3, wherein the HVR-H1 and / or HVR-H2 are selected from the group consisting of HVR-H1 sequences of the disclosure (e.g., X1TFX2X3YX4IHWV, where 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, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); etc. and SEQ ID NOs: 1-52 and 137-158), and HVR-H2 sequences of the disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 202); IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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, where 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, where 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, where X1 is K or R, X2 is A or T, and X3 is D or 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 amino acid sequences selected from SEQ ID NOs: 53 to 136 and 159 to 164. ;

[0118] In some embodiments, at least one of HVR-H1, HVR-H2, and HVR-H3 of an antibody heavy chain variable region adopts multiple conformations, which in some embodiments can be assessed or detected using techniques known in the art, including, but not limited to, structure determination (e.g., X-ray crystallography or NMR) and / or computer modeling.

[0119] A polynucleotide encoding a set of antibody light and / or heavy chain variable regions can be cloned into any vector suitable for expression of a portion or the entirety of the light or heavy chain sequence. In some embodiments, the polynucleotide cloned into a vector allows for the generation of a portion or the entirety of the light or heavy chain sequence fused to all or a portion of a viral coat protein (i.e., generating a fusion protein) and displayed on the surface of a particle or cell. Several types of vectors are available and can be used to practice the present disclosure, such as, for example, phagemid vectors. Phagemid vectors generally contain various components, including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components known to those skilled in the art. In some embodiments, a polynucleotide encoding a set of antibody light and / or heavy chain variable regions can be cloned into a vector for expression in bacterial cells for bacterial display or yeast cells for yeast display. Exemplary vectors are described in U.S. Patent Application Publication No. US20160145604. In some embodiments, the vector is a display vector that includes, from 5' to 3', a polynucleotide encoding an amino acid sequence to be displayed on a surface (e.g., the surface of a phage, bacteria, yeast, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide capable of display 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 adaptor, or a truncated or derivative thereof. In certain embodiments, the second polynucleotide is gene III of filamentous phage M13, or a truncated or derivative thereof. In some embodiments, the surface peptide is for phage display, yeast display, bacterial display, or mammalian display, or shuttling display across these. In some embodiments, the amino acid sequence and the surface peptide, when expressed, are displayed on the surface as a fusion protein.In some embodiments, the vector further comprises a fusion tag 5' to the first restriction site or 3' to the second restriction site.

[0120] Certain aspects of the present disclosure relate to populations of cells containing the vector(s) described herein. The light and / or heavy chains of antibodies encoded by polynucleotides generated by any of the techniques described herein or other suitable techniques can be expressed and screened to identify antibodies with the desired structure and / or activity. Expression of antibodies can be performed, 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, yeast, or mammalian cells. Methods for transfecting bacterial, yeast, or mammalian cells are known in the art and described in the references cited herein. Expression of polypeptides (e.g., antibody chains) in these cell types (e.g., expression from the libraries of the present disclosure) and screening for antibodies of interest are described in more detail below.

[0121] 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 by secretion into the medium and / or into the bacterial cytoplasm as described by Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, the V H and V LThe single domains encoding are each linked 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). Fusions of these genes are assembled into dicistronic constructs, so that they can be expressed from a single vector and secreted into the periplasm of E. coli where they can be refolded and recovered in active form (Skerra et al., Biotechnology, 1991, 9:273). For example, an antibody heavy chain gene can be expressed along with an antibody light chain gene to produce an antibody or antibody fragment.

[0122] In other embodiments, the antibody sequences are expressed on the membrane surface of a prokaryote, such as E. coli, using a secretion signal and lipidation moieties, e.g., as described in US20040072740; US20030100023; and US20030036092.

[0123] Alternatively, antibodies can be expressed and screened by anchored periplasmic expression (APEx2-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.

[0124] Higher eukaryotic cells, such as mammalian cells, e.g., myeloma cells (e.g., NS / 0 cells), hybridoma cells, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells, can also be used to express the antibodies of the present disclosure. Typically, antibodies expressed in mammalian cells are engineered to be secreted into the medium or expressed on the surface of the cell. The antibodies or antibody fragments can be expressed, for example, as intact antibody molecules or as individual V proteins. H and V LIt can be produced as fragments, Fab fragments, single domains, or as single chains (scFv).

[0125] In other embodiments, antibodies can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103:9637). In some embodiments exemplified above and below, antibodies can be selected after some or all of the light or heavy chain sequences are generated, e.g., using phage display, fused to all or a portion of a virus-encoded protein (i.e., generating a fusion protein) and displayed on the surface of a particle or cell.

[0126] Certain aspects of the present disclosure relate to non-human animals that comprise the polynucleotide library of the present disclosure. For example, the non-human animals of the present disclosure can be modified such that their genome comprises a polynucleotide encoding the heavy chain variable region of the present disclosure. In a non-limiting example, a transgenic mouse is produced that comprises 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., a mouse) expresses an antibody or heavy chain encoded by a polynucleotide. Techniques for modifying one or more immunoglobulin loci of a non-human animal are known in the art (e.g., the method used to produce Xenomouse™).

[0127] Screening of antibodies from the library of the present disclosure can be performed by any suitable means known in the art. For example, binding activity can be assessed by standard immunoassays and / or affinity chromatography. Screening of the antibodies of the present disclosure for catalytic function (e.g., proteolytic function) can be accomplished using standard assays, such as hemoglobin plaque assays. Determination of the binding affinity of the antibody to the target can be assayed in vitro using a variety of known techniques, such as Bio-Layer Interferometry (BLI), as exemplified below, using a BIACORE™ instrument that measures the binding rate of an antibody to a given target or antigen based on surface plasmon resonance, or the ForteBio Octet® RED96 platform (Pall Life Sciences). In vivo assays can be performed by using any of a number of animal models, and then tested in humans, if desired. Cell-based biological assays are also contemplated. Antibodies or antigen-binding fragments can be further selected for functional activity, such as antagonist or agonist activity. Exemplary screening methods are described herein. For example, in some embodiments, the binding affinity between the fab fragment(s) and one or more target(s) is measured using BLI by tagging the antigen with a human IgG1-Fc tag and capturing it with an Anti-hIgG-Fc Capture (AHC) Biosensor. The Fab can be tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in a host cell such as E. coli, and purified, for example, using Ni-NTA resin. The affinity can then be measured using an AHC sensor (anti-human IgG-Fc capture dip and read biosensor) immersed in a well containing purified fab diluted, for example, to 5-10 μg / mL in kinetics buffer.

[0128] After identifying binders by binding to a target or antigen and / or functional assays, the nucleic acids can be extracted. The extracted DNA can then be used directly to transform E. coli host cells, or the coding sequences can be amplified, for example, using PCR with suitable primers and sequenced by any typical sequencing method. The variable domain DNA of the binders can be digested with restriction enzymes and then inserted into a vector for protein expression.

[0129] IV. Antibodies and Antibody Production Provided herein are antibodies defined and selected from the libraries described herein. Certain aspects of the disclosure relate to antibody light or heavy chain HVRs, variable regions comprising HVRs and / or polynucleotide(s) encoding same. In some embodiments, the HVRs and / or variable regions are part of an antibody fragment, a full-length antibody, or a single chain variable fragment (scFv).

[0130] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula I) X1TFX2X3YX4IHWV, where 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, where 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, where 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, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 is selected from the group consisting of: (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where 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) IGX1IYX2SGX3TX 4YNPSLKSRV, where 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, where 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, where 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, where 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, where 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, HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is of formula I: X1TFX2X3YX4IHWV, where 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); or of formula II: YSIX1SGX2X3WX4WI, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, and X4 is A, G, S, or T (SEQ ID NO: 201). is D or G, X5 is R, S, or Y, and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) IGX1INPNX2GX3TX4YAQKF QGRV, where 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, where 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 heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is a formula selected from the group consisting of: (Formula I) X1TFX2X3YX4IHWV, where 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, where 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, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200). HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XII) IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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).

[0131] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula X1TFX2X3YX4IHWV, where 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 (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, and X5 is R, S, or Y. and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) VSX1ISGX2GX3X4T YYADSVKGRF, where 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, where 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, where X1 is I, and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, where 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).

[0132] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula X1TFX2X3YX4IHWV, where 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 XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XII) IGX1IYX2SG X3TX4YNPSLKSRV, where 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, where 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).

[0133] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula YSIX1SGX2X3WX4WI, where 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 IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, and X5 is R, S, or Y. and X6 is P or T) (SEQ ID NO: 201); (Formula V) IGX1IX2X3SGSTYYSPSLKSRV, where 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, where 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) VSX1ISGX2GX3X4 TYYADSVKGRF, where 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, where 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, where X1 is I, and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF, where 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).

[0134] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula YSIX1SGX2X3WX4WI, where 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); (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XII) IGX1IYX2S and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: (Formula XII)GX3TX4YNPSLKSRV, where 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, where 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).

[0135] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula FSLSTX1GVX2VX3WI, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); and (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where 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, where 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) VSX1ISGX2GX3X4TYYADSVK GRF, where 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, where 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, where X1 is I, R, or X1 is 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, where 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).

[0136] In some embodiments, the heavy chain variable region comprises an HVR-H1 comprising an amino acid sequence according to the formula FSLSTX1GVX2VX3WI, where X1 is G or S, X2 is A or G, and X3 is A, G, S, or T (SEQ ID NO: 200); (Formula XI) IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); (Formula XII) IGX1IYX2SGX3TX4YN and an HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of: PSLKSRV, where 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, where 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).

[0137] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and / or HVR-H2 comprise the amino acid sequences listed in Table 1 below. Table 1: Heavy chain HVR sequence TIFF2024178314000002.tif250170TIFF2024178314000003.tif251170TIFF2024178314 000004.tif251170TIFF2024178314000005.tif251170TIFF2024178314000006.tif86170

[0138] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, which 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.

[0139] In some embodiments, provided herein is an antibody heavy chain having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein the HVR-H1 and / or HVR-H2 is any of the HVR-H1 and / or HVR-H2 described herein. In some embodiments, the HVR-H1 comprises an amino acid sequence selected from any HVR-H1 sequence of the disclosure (e.g., X1TFX2X3YX4IHWV, where 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, where 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, where 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 is any HVR-H2 of the disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where 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, where 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, where 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, where X1 is A, D, or E, and X2 is K or N (SEQ ID NO: 208); 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 amino acid sequences selected from SEQ ID NOs: 53 to 136 and 159 to 164.

[0140] In some embodiments, provided herein is an antibody heavy chain having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52.

[0141] In some embodiments, provided herein is an antibody heavy chain having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136.

[0142] In some embodiments, provided herein is an antibody heavy chain having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of Formula (I), Formula (II), and Formula (III), or wherein 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 having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, or wherein HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, 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 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, 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 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, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 10, 17, 29, 36, and 52, or 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.

[0143] In some embodiments, provided herein is an antibody heavy chain having a heavy chain variable region comprising 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.

[0144] In certain embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, and the HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (II) HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VI); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VII); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VIII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (V); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (V);In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XI); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XII); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (XI); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IV); HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (IX); HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (X); HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (VIII); and HVR-H1 comprising the amino acid sequence of formula (III) and HVR-H2 comprising the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising an amino acid sequence of formula (II) and HVR-H2 comprising an amino acid sequence of formula (XIII); HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (XIII); and HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (XIII).

[0145] The heavy chain HVR sequences described herein may be included in any combination in the antibody heavy chain or heavy chain variable region of the present disclosure. In some embodiments, the heavy chain variable region includes HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, the heavy chain variable region includes HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, the heavy chain variable region includes HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, the heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region comprises HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.In some embodiments, the heavy chain variable region comprises HVR-H1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, HVR-H2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.

[0146] In certain embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 1 and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 122; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprises the amino acid sequence of SEQ ID NO: 154 and HVR-H2 comprise the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 145 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 128; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63 HVR-H1 comprising the amino acid sequence of SEQ ID NO: 153 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 67; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 100; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 123 VR-H2; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 126; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 129; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 124; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 130;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 150 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 132; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 82; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 149 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 comprising the amino acid sequence of SEQ ID NO:26 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:151 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:53; HVR-H1 comprising the amino acid sequence of SEQ ID NO:34 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:162; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:104; HVR-H1 comprising the amino acid sequence of SEQ ID NO:5 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:6 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:116. HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:121; HVR-H1 comprising the amino acid sequence of SEQ ID NO:17 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:101; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:114; HVR-H1 comprising the amino acid sequence of SEQ ID NO:29 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:112; HVR-H1 comprising the amino acid sequence of SEQ ID NO:152 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:63; HVR-H1 comprising the amino acid sequence of SEQ ID NO:156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 48 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 50 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 163; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 160; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:87; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:92; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:93; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:97; HVR-H1 comprising the amino acid sequence of SEQ ID NO:158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:103; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 164; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 137 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 3 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 85; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 120; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 140 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 131; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 141 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 142 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 159; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 143 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 116; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 144 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 146 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 133; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 148 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118.

[0147] In some embodiments, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, where HVR-H1 and HVR-H2 are listed in Table 1. In some embodiments, HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, the 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.

[0148] In some embodiments, the heavy chain variable region further comprises variable region heavy chain framework sequences located adjacent to and between the HVRs according to the following 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 are: FW-H1 is EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 165), FW-H2 is RQAPGKGLEW (SEQ ID NO: 166); FW-H3 is TISRDSNSKNTLYLQLNSLRAEDTAVYYC (SEQ ID NO: 167); FW-H4 is WGQGTLVTVSS (sequence number 168).

[0149] In some embodiments, the heavy chain variable region comprises an alternative 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 are FW-H1 of SEQ ID NO: 165, FW-H2 of SEQ ID NO: 166, SEQ ID NO: 167 or FW-H3 with an arginine to lysine mutation at R19, and FW-H4 of SEQ ID NO: 168.

[0150] In some embodiments, further provided herein is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region of the present disclosure and the light chain comprises any light chain variable region known in the art (e.g., including HVR-L1, HVR-L2, and HVR-L3). In some embodiments, the antibody light chain variable region comprises 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 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 HVR-L1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-264, and 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 patent applications filed with Attorney Docket Nos. 69540-3000100, 69540-2000140, and / or 69540-2000100, the disclosures of which are each incorporated 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 patent applications filed with Attorney Docket Nos. 69540-3000100, 69540-2000140, and / or 69540-2000100, the disclosures of which are each incorporated by reference in their entireties.

[0151] 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 for similar applications are being explored. Several protein families with non-Ig structures such as protein A, fibronectin, ankyrin repeats, adnectins, affibodies, anticalins, DARPins, engineered Kunitz inhibitors or lipocalins, cyclic and polycyclic peptides can be endowed with new binding sites by employing combinatorial engineering methods such as site-directed random mutagenesis combined with phage display, yeast display, or other molecular selection techniques. These new alternative binding reagents are collectively referred to as engineered protein scaffolds, and refer to the fact that rigid natural protein structures are used to modify existing binding sites or to endow new binding sites for a given target using the dynamic binding motifs or units introduced herein. These protein scaffolds often offer practical advantages, including improved stability and high production yields in microbial expression systems, compared to antibodies or their recombinant fragments. These novel binding proteins are obtained by biomolecular engineering processes to achieve tight target binding activity, and can also be subjected to further selection schemes focusing on other desired properties (e.g., solubility, thermostability, protease resistance, etc.). As a result, engineered protein scaffolds have become attractive for many applications in biotechnology and biomedical research, especially for multispecific binding motifs. Efforts to generate such alternative binding proteins with beneficial properties are directed towards therapeutic uses, with particular emphasis on the structure and function of the biomolecule, as well as approaches towards clinical applications.

[0152] In some embodiments, further provided herein are one or more polypeptides (e.g., scaffold polypeptides 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 repeats, adnectins, affibodies, anticalins, DARPins, engineered Kunitz inhibitors or lipocalins, cyclic and polycyclic peptides) that comprise 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 disclosure (e.g., X1TFX2X3YX4IHWV, where 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, where 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, where 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 is any HVR-H2 of the disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL, where 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, where X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 202); IGX1IYX2SGX3TX4YNPSLKSRV, where 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, where 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, where 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, where 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, where X1 is K or R, X2 is A or T, and X3 is D or Y. and X4 is G or Y, X5 is D or E, and X6 is P or S (SEQ ID NO: 207); IGX1IX2X3SGSTYYSPSLKSRV, where X1 is A, D, or E, X2 is S or Y, and X3 is H or Y (SEQ ID NO: 208); IGX1IYX2SGX3TX4YNPSLKSRV, where X1 is D, E, or S, X2 is H or Y, and X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF, where 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 a HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 53-136 and 159-164. In some embodiments, the polypeptide comprises a HVR-H3 comprising an amino acid sequence selected from any HVR-H3 sequence of the disclosure (e.g., SEQ ID NOs: 223-256). In some embodiments, the polypeptide comprises a HVR-L1 comprising an amino acid sequence selected from any HVR-L1 sequence of the disclosure (e.g., SEQ ID NOs: 257-264). In some embodiments, the polypeptide comprises a HVR-L3 comprising an amino acid sequence selected from any HVR-L3 sequence of the disclosure (e.g., SEQ ID NOs: 265-274).

[0153] In some embodiments, a 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, a polypeptide comprises two of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and / or HVR-L3 sequences described herein, the two being HVR-H1 and HVR-H2; HVR-H1 and HVR-H3; HVR-H1 and HVR-L1; HVR-H1 and HVR-L3; HVR-H2 and HVR-H3; HVR-H2 and HVR-L1; HVR-H2 and HVR-L3; HVR-H3 and HVR-L1; HVR-H3 and HVR-L3; or HVR-L1 and 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 HVR-H1, HVR-H2, and HVR-H3; HVR-H1, HVR-H2, and HVR-L1; HVR-H1, HVR-H2, and HVR-L3; HVR-H1, HVR-H3, and HVR-L1; HVR-H1, HVR-H3, and HVR-L3; HVR-H1, HVR-L1 and HVR-L3; HVR-H2, HVR-H3, and HVR-L1; HVR-H2, HVR-H3, and HVR-L3; HVR-H2, HVR-L1, and HVR-L3; or HVR-H3, HVR-L1, and 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 HVR-H1, HVR-H2, HVR-H3, and HVR-L1; HVR-H1, HVR-H2, HVR-H3, and HVR-L3; HVR-H1, HVR-H2, HVR-L1, and HVR-L3; HVR-H1, HVR-H3, HVR-L1, and HVR-L3; or HVR-H2, HVR-H3, HVR-L1, and 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, the five being HVR-H1, HVR-H2, HVR-H3, HVR-L1, and HVR-L3.

[0154] Further provided herein, in some embodiments, is an antibody fragment or scFv comprising a light chain variable region and a heavy chain variable region of the disclosure.

[0155] In some embodiments, an antibody or antibody fragment of the present disclosure binds to at least one target (e.g., a target protein or target epitope) or at least two targets with a specific binding affinity. For example, in some embodiments, an antibody or antibody fragment of the present disclosure binds to at least about 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 In some embodiments, the antibody or antibody fragment of the present disclosure binds to at least one target or at least two targets with an equilibrium dissociation constant (Kd) of about 10 -7 ~about 10 -11 It binds to at least one target or at least two targets with an equilibrium dissociation constant (Kd) of M. Exemplary assays for determining binding affinity are described and exemplified below (see, e.g., ForteBio assay in Example 4 below).

[0156] In some embodiments, the antibody or antibody fragment of the present disclosure has a melting temperature (Tm) of at least 60°C. For example, in some embodiments, the antibody or antibody fragment of the present disclosure has a Tm of about 60°C to about 90°C, about 65°C to about 90°C, about 70°C to about 90°C, about 75°C to about 90°C, about 80°C to about 90°C, about 85°C to 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, the antibody or antibody fragment of the present disclosure has a Tm of about 60°C to about 90°C. Various methods for measuring the Tm of an antibody or antibody fragment are known in the art. Exemplary assays for determining the Tm of an antibody are described and exemplified below (see, for example, the DSF assay in Example 4 below).

[0157] Antibodies of the present disclosure can be produced using recombinant methods and compositions described, for example, in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids are provided that encode any of the antibodies described herein. Such nucleic acids can be used to encode the V L and / or V H In some embodiments, provided herein is one or more vectors (e.g., expression vectors) comprising such a nucleic acid. In some embodiments, provided herein is a host cell comprising such a nucleic acid. In one such embodiment, the host cell comprises: (1) an amino acid sequence comprising the V of the antibody; L Amino acid sequence containing the V of the antibody H or (2) a vector comprising a nucleic acid encoding an amino acid sequence comprising the V L A first vector comprising a nucleic acid encoding an amino acid sequence comprising Hand a second vector comprising (e.g., transformed with) a nucleic acid encoding an amino acid sequence comprising:

[0158] To recombinantly produce an antibody of the present disclosure, for example, a nucleic acid encoding the above-described antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0159] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. 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 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254 (for a description of the expression of antibody fragments in E. coli)). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0160] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0161] Suitable host cells for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used for transfection of insect cells, particularly Spodoptera frugiperda cells.

[0162] Plant cell cultures can also be used as hosts, see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0163] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 cells or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, for example, 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 hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); see, for example, Mather et al., Annals TRI cells, as described in NYAcad.Sci.383:44-68(1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216(1980)); and myeloma cell lines, such as Y0, NS0 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 (BKC Lo,ed.,Humana Press,Totowa,NJ),pp.255-268(2003).

[0164] Bispecific antibodies with identical / shared / single heavy chains Further provided herein are bispecific antibodies having identical heavy chain variable regions 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, the first light chain being kappa C Ldomain (e.g., human kappa C L domain), and the second light chain contains lambda C L domain (e.g., human lambda C L domain). Kappa C L Domain and Lambda C L Methods for making and / or purifying bispecific antibodies comprising a) two identical heavy chain variable regions (e.g., any one of the heavy chain variable regions described herein) and b) a first light chain variable region and a kappa C 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) and b) a first light chain variable region and a kappa C domain. L a first light chain comprising a second light chain variable region and a lambda C domain; and L A second light chain containing a domain (e.g., kappa C L The second light chain constant region containing the domain is lambda C L Bispecific antibodies can be constructed and expressed (e.g., cloned into one or more expression vectors and expressed in one or more suitable host cells) that contain a bispecific IgG (e.g., a IgG1 domain with both kappa and lambda replaced by a C domain with ... L Bispecific IgG containing the IgG1 domain can be purified using the following steps: First, total IgG is recovered from the culture supernatant using Protein A or IgG-CH1 CaptureSelect affinity chromatography, which removes free light chains and other contaminants. Next, kappa C is isolated using KappaSelect affinity resin. L Capture IgG containing the Lambda C domain L Monospecific IgGs containing light chains containing only the domain are removed in the column flow-through. Finally, the LambdaFabSelect affinity resin is used to recover the pure bispecific kappa-lambda bodies and the kappa C that does not bind to the resin. LAlternatively, bispecific common heavy chain IgG (e.g., IgG as described above) can be purified by Protein A and each light chain C can be separated based on one or more differences in the biophysical properties of the different light chains (e.g., different molecular weights, different isoelectric points (pI), etc.). L The separation can be achieved using domain specific resins.

[0165] In some embodiments, a bispecific antibody comprises two antibody light chain variable regions and two identical heavy chain variable regions, the bispecific antibody comprises 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, the second antibody heavy chain variable region having an identical sequence 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 embodiments, the first and second binding domains bind to different epitopes on the same biomolecule. In some embodiments, the first antibody heavy chain variable region is a portion of a first antibody heavy chain that comprises a first heavy chain variable region and a first heavy chain constant region (e.g., including CH1, hinge, CH2, and CH3). In some embodiments, the second antibody heavy chain variable region is a portion of a second antibody heavy chain comprising a 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 a portion of a first antibody light chain comprising a first light chain variable region and a first light chain constant region. In some embodiments, the second antibody light chain variable region is a portion of a second antibody light chain comprising a second light chain variable region and a second light chain constant region. In some embodiments, the first and second antibody heavy chains have sequences identical to the heavy chains of the present disclosure.

[0166] Further provided herein are methods of making a bispecific antibody having identical heavy chain variable regions 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) generating a 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 disclosure.

[0167] In some embodiments, the bispecific antibodies described herein may have additional specificities, for example, one of the antigen-binding or target-binding sites of the bispecific antibody may specifically bind to more than one target.

[0168] Methods for making / producing bispecific antibodies are known in the art. The production of full-length bispecific antibodies can be based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography steps, which are quite cumbersome and give low product yields. Similar techniques are disclosed in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0169] V.Kit 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, including 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 transfecting one or more of the polynucleotides into a host cell. EXAMPLES

[0170] The present disclosure will be more fully understood by referring to the following examples. However, the examples should not be interpreted as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in consideration thereof will occur to those skilled in the art, and are within the spirit and scope of the present application and the scope of the appended claims.

[0171] Example 1: Identification of a minimal set of dynamic motifs in hypervariable regions To understand the variability of antibody variable domains at the structural level, we developed an algorithm that maps the geometric alignment of antibody variable domains and further calculates the structural and sequence entropy based on the geometric alignment. Taking such an approach, we use the classical theory of antibody diversity determined by the well-established V(D)J gene rearrangement process in combination with conformational diversity by dynamic units (template-directed conformational selection by Linus Pauling; see, e.g., James, L. and Tawfik, D. “Conformational diversity and protein evolution-a 60-year-old hypothesis revisited”, Trends Biochem Sci. 2003 Jul;28(7):361-8), allowing the sampling of a nearly infinite epitope space by selecting and adapting antibody binding sites. As an example, we used the algorithm to analyze the structural and sequence variability for high-resolution crystal structures of 113 human antibody variable heavy chain domains. For every position in the variable heavy chain domain, the entropy was calculated and plotted (Figure 1A; structural entropy is the bold line, sequence entropy is the dotted line). The results obtained by the geometric alignment-based structural and sequence entropy calculations were used to identify hypervariable (HVR) regions and to identify important positions on these variable regions. For comparison, the HVRs (as defined by the method described above) and CDRs (as defined by Kabat) of an exemplary antibody heavy chain variable domain sequence were identified (Figure 1B).

[0172] Interestingly, the variability assessed by structural alignment was generally lower than that observed by sequence alignment. Although the variability was generally low when assessed by structural alignment, there were many sites / regions with dramatic structural changes, suggesting that these variable sites may play important roles in antibody function. Furthermore, some of these hypervariable regions had multiple conformations and showed high flexibility. The identification of highly variable residue regions provided a more comprehensive picture of the conservation and variability of antibody variable domains that can be exploited for novel antibody design. The identification of this dynamic motif allowed for a wide range of structural diversity to be covered with a small number of amino acid sequences. A surprising advantage of this approach to antibody design was that a more limited number of dynamic motifs could be utilized in the variable regions to cover a wide range of antibody structural diversity, providing these antibodies with a wide range of flexibility that may enable binding to multiple antigens of interest. Thus, the dynamic heavy chain library was constructed using a single human germline or human germline-derived sequence for the invariant residues, but a limited number of dynamic motifs (10 6 , 10 10 or higher) captures the extensive structural variability identified in these two regions.

[0173] Example 2: Construction of a consensus heavy chain library Heavy chain library construction To begin construction of the heavy chain library, for the variable region HVR-H1, 3 groups of degenerate oligos were designed based on the formula shown in Table 2, resulting in 112 unique HVR-H1 sequences. For the variable region HVR-H2, 7 groups of degenerate oligos were designed based on the formula shown in Table 2, resulting in 565 unique HVR-H2 sequences. The synthesized degenerate oligos were converted to double-stranded DNA by the following protocol: 0.75 μL of 0.2 μM template oligo was mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mix, 1 μL of 100 μM forward primer, 1 μL of 100 μM reverse primer, 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and 33 μL of water. The PCR solution was preheated to 96° C. for 5 minutes, then 14 cycles were performed (15 sec at 96° C., 15 sec at 60° C., 6 sec at 72° C.), followed by a 3 min extension at 72° C. VH_vr1 was amplified using primer pair F_1999 (CGTTTGTCCTGTGCAGCTTCCGG) (SEQ ID NO: 211) and R_1999 (CGAGGCCCTTACCCGGGGCCTGACG) (SEQ ID NO: 212), while VH_vr2 was amplified using primer pair F_2003 (CCGGGTAAGGGCCTCGAGTGG) (SEQ ID NO: 213) and R_2003 (GAGCACGTCCGTTCGAATTGTCGCGACTTATAG) (SEQ ID NO: 214).

[0174] The double stranded VH_vr1 and VH_vr2 were joined via overlapping sequences at their 5' or 3' ends. The protocol used was as follows: 20 ng VH_vr1 and 20 ng VH_vr2 templates were mixed with 10 μL 5x PrimeSTAR buffer, 4 μL dNTP mix, 1 μL 100 μM F_1999 primer, 1 μL 100 μM R_2003 primer, 0.5 μL PrimeSTAR HS DNA Polymerase (2.5 U / μL) and water (up to 50 μL), the mixture was preheated at 96°C for 5 min, then 14 cycles (96°C for 15 s, 60°C for 15 s, 72°C for 10 s) were performed, followed by extension at 72°C for 3 min. These PCR fragments were then purified by gel electrophoresis (GENEray Gel Extraction kit), digested with BspEI and BstBI (Thermo Scientific), and then cloned into the filter vector FTV014 digested with the same two enzymes. The ligation mixture was used to transform DH10B cells by electroporation, and a number of colonies exceeding 10-fold the calculated diversity were recovered for plasmid preparation. The purified plasmids constituted library VH-vr12. Table 2: Formulas of HVR-H1 and HVR-H2 designed variant sequences. TIFF2024178314000007.tif244170TIFF2024178314000008.tif33170

[0175] The following protocol allows 5Several hundred degenerate oligos encoding VH_vr3 with sequence diversity close to that of the original VH_vr3 were designed, synthesized, and converted to double-stranded DNA. 0.75 μL of 0.2 μM template oligo was mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mix, 1 μL of 100 μM forward primer, 1 μL of 100 μM reverse primer, 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and 33 μL of water. The PCR solution was preheated at 96°C for 5 min, then 14 cycles (96°C for 15 s, 60°C for 15 s, 72°C for 6 s) were performed, followed by extension at 72°C for 3 min. The forward primer was S1089 (ACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTG) (SEQ ID NO: 215) and the reverse primer was S1090 (GAGGAGACGGTGACTAGTGTTCCTTGACCCCA) (SEQ ID NO: 216). The resulting synthetic DNA was then purified by gel electrophoresis (GENEray Gel Extraction kit), digested with AflII and SpeI (Thermo Scientific), and then cloned into the filter vector FTV012 digested with the same two restriction enzymes. The ligation mixture was used to transform DH10B cells by electroporation, and a number of colonies exceeding 10-fold the calculated diversity was recovered for plasmid preparation. The purified plasmids constituted library VH-vr3.

[0176] To assemble the full-length VH library, the purified VH-vr3 library plasmid mixture was digested with AflII and SpeI (NEB) and the vr3 coding fragment was purified by gel electrophoresis (GENEray Gel Extraction kit) and cloned into the VH-vr12 library plasmid mixture digested with the same two restriction enzymes. After desalting the ligation products (QIAquick® PCR Purification Kit (QIAGEN)), rolling circle amplification (RCA) was performed. RCA was performed as follows: 40 ng of ligation product was mixed with 10 μL of 10x NEBuffer4, 50 μL of 100 μM pd(N)8, and water (up to 88.5 μL), heated to 95°C for 3 minutes, and annealed for 65 cycles (30 seconds each cycle) with 1°C decrease per cycle. After adding 10 μL of 10 mM dNTP mix, 1 μL of 100×BSA, and 0.5 μL of Phi29 DNA polymerase, the annealed reaction was incubated overnight at 30° C. The RCA products were first digested with NotI, and the DNA fragments were purified (QIAquick® PCR Purification Kit) and further digested with XhoI. The digested products were then ligated with T4 DNA ligase (Thermo Scientific). After purification by ethanol precipitation, the ligation products were used to transform DH10B cells by electroporation. The purified plasmids constituted library VH-vr123. Each of these constructs shared the same framework region, i.e., FW-H1 (SEQ ID NO: 165), FW-H2 (SEQ ID NO: 166), FW-H3 (SEQ ID NO: 167), and FW-H4 (SEQ ID NO: 168).

[0177] The plasmid mixture of the two heavy chain libraries was digested with PvuI and Acc65I and ligated into the phagemid vector Fad40, which was also digested with the same two restriction enzymes. The ligation mixture was used to transform DH10B cells, and the resulting library was purified, quantified, and stored for assembly of the complete phagemid library.

[0178] Construction of VL library To begin the construction of the light chain library, 18 groups of degenerate oligos and 5 predefined oligos were designed for each of the variable regions VL_vr1 and VL_vr2. These were converted to double-stranded DNA by the following protocol: 0.75 μL of 0.2 μM template oligo was mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mix, 1 μL of 100 μM forward primer, 1 μL of 100 μM reverse primer, 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and 33 μL of water. The PCR solution was preheated at 96°C for 5 min, then 14 cycles (96°C for 15 s, 60°C for 15 s, 72°C for 6 s) were performed, followed by extension at 72°C for 3 min. VL_vr1 was amplified using primer pair F_2898 (TACTTATGTAGGCGATCGGGTCACCATCACCTGC) (SEQ ID NO: 217) and R_2898 (CGGAGCTTTTCCTGGTTTCTGTTGATAC) (SEQ ID NO: 218), while VL_vr2 was amplified using primer pair F_2013 (GAAACCAGGAAAAGCTCCGAAG) (SEQ ID NO: 219) and R_2013 (CGTCCCGGAACCGGATCCAGAGAAGCGAG) (SEQ ID NO: 220).

[0179] The double stranded VL_vr1 and VL_vr2 were joined via overlapping sequences at their 5' or 3' ends. The protocol used was as follows: 20 ng VL_vr1 and 20 ng VL_vr2 templates were mixed with 10 μL 5x PrimeSTAR buffer, 4 μL dNTP mix, 1 μL 100 μM F_2898 primer, 1 μL 100 μM R_2013 primer, 0.5 μL PrimeSTAR HS DNA Polymerase (2.5 U / μL) and water (up to 50 μL), the mixture was preheated at 96°C for 5 min, then 14 cycles (96°C for 15 s, 60°C for 15 s, 72°C for 10 s) were performed, followed by extension at 72°C for 3 min. These PCR fragments were then purified by gel electrophoresis (GENEray Gel Extraction kit), digested with PvuI and BamHI (Thermo Scientific), and then cloned into the filter vector FTV015 digested with the same two enzymes. The ligation mixture was used to transform DH10B cells by electroporation, and a number of colonies exceeding 10-fold the calculated diversity was recovered for plasmid preparation. The purified plasmids constituted library VL-vr12.

[0180] Twenty-two groups of degenerate oligos encoding VL_vr3 were designed, synthesized, and converted to double-stranded DNA by the following protocol: 0.75 μL of 0.2 μM template oligo was mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mixture, 1 μL of 100 μM forward primer F2929 (ACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAAC) (SEQ ID NO: 221), 1 μL of 100 μM reverse primer R2929 (GATCTCCACCTTGGTACCCTGTCCGAA) (SEQ ID NO: 222), 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and 33 μL of water. The PCR solution was preheated at 96°C for 5 minutes, then 14 cycles (96°C for 15 seconds, 60°C for 15 seconds, 72°C for 6 seconds) were performed, followed by extension at 72°C for 3 minutes. The double-stranded DNA encoding VL_vr3 was then purified by gel electrophoresis (GENEray Gel Extraction kit), digested with PstI and Acc65I (Thermo Scientific), and then cloned into the filter vector FTV013 digested with the same two restriction enzymes. The ligation mixture was used to transform DH10B cells by electroporation, and a number of colonies exceeding 10-fold the calculated diversity was recovered for plasmid preparation. The purified plasmids constituted library VL-vr3.

[0181] To assemble the full-length VL library, the purified VL-vr3 library plasmid mixture was digested with PstI and Acc65I (NEB), and the vr3 coding fragment was purified by gel electrophoresis (GENEray Gel Extraction kit) and then cloned into the VL-vr12 library plasmid mixture digested with the same two restriction enzymes. The ligation product was used to transform DH10B cells by electroporation, and a number of colonies more than 10 times the calculated diversity was recovered for plasmid preparation. The purified plasmid constituted library VL-vr123. The vr123 insert of library plasmid VL-vr123 was then transferred to the phagemid vector Fad40 using the restriction enzymes PvuI and Acc65I. The size of the library containing Fad40-vr123 was 4*10 7 reached.

[0182] Building a complete dynamic library The dynamic library consisted of a heavy chain library derived from the VH-vr123 library and a light chain library derived from the Fad40-vr123 library. Both the VH-vr123 and Fad40-vr123 library plasmids were digested with BspEI and SpeI (Thermo Scientific). The DNA fragments encoding the heavy chains derived from the VH-vr123 library were cloned into the vector backbone derived from the Fad40-vr123 library. The ligation products were desalted (QIAquick® PCR Purification Kit (QIAGEN)) prior to rolling circle amplification (RCA). RCA was performed as follows: 40ng of the ligation product was mixed with 10μL of 10x NEBuffer4, 50μL of 100μM pd(N)8, and water (maximum 88.5μL), heated at 95℃ for 3 minutes, and annealed for 65 cycles (30 seconds each cycle) with 1℃ step. After adding 10μL of 10mM dNTP mix, 1μL of 100xBSA, and 0.5μL of Phi29 DNA polymerase, the annealed reaction was incubated at 30℃ overnight. The RCA product was first digested with NotI, and the DNA fragment was purified (QIAquick® PCR Purification Kit) and further digested with Acc65I. The digested product was then ligated with T4 DNA ligase (Thermo Scientific). After purification by ethanol precipitation, the ligation product was transformed into ER2738 cells by electroporation. A total of 1.4*10 10 Colonies were picked (2xYT, 1% glucose, 100 μg / mL ampicillin) to generate the DPL6 library.

[0183] Example 3: Screening of a consensus heavy chain library to isolate antibodies of interest Preparation of dynamic library phagemid particles To prepare the dynamic library phagemid particles for antigen panning, 5.0 liters of ER2738 cells (described in Example 2 above) containing the dynamic library were cultured at a starting OD of 0.1. 600 The cultures were inoculated into a medium containing 2xYT, 2% glucose, 100 μg / mL ampicillin, and 12.5 μg / mL tetracycline at 100°C for 24 hours. The cultures were grown to an OD of 0.6–0.8 with shaking at 250 rpm. 600 The cells were then grown at 37°C until the OD reached 1.0. The cells were then infected with M13KO7 helper phage at a multiplicity of infection (MOI) of 10 for 30 min at 37°C. The infected ER2738 cells were grown overnight at 22°C in 3.2 liters of medium containing 2xYT, 100 μg / mL ampicillin and 50 μg / mL kanamycin. The culture supernatant was then harvested by centrifugation at 10,000 rpm for 15 min and filtered through a 0.45 μm low-binding membrane filter (Corning). Phagemid particles were then precipitated from the filtered supernatant using PEG / NaCl and resuspended in PBS. An additional round of precipitation with PEG / NaCl followed by resuspension in PBS was performed. Phage concentrations were determined by OD 268 Measurement of OD 268 One unit is approximately 1*10 13 The total number of phagemid particles was determined by 100 μg / mL (assuming phage particles / mL) and confirmed by plaque assay. Library phagemid particles were stored at -80°C in 20% glycerol.

[0184] Phage library panning Antigen proteins at concentrations of 1-30 μg / ml were coated onto Maxisorp strips (Thermo Scientific, Cat. No. 446469) overnight at 4°C. Multiple antigen wells 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 μL / well of phagemid particle solution (typically 1-5*10 in 2% milk in PBS) was added. 12phages) were added to four parallel wells and incubated for 1-2 h. The wells were then washed several times with PBS containing increasing concentrations of Tween 20 (0.1% to 0.3%) and finally with PBS alone. Bound phagemid particles were eluted from the wells with 100 μL of 0.2 M glycine-HCl for 10 min at room temperature. Eluted phages were immediately neutralized with 18 μL of 1 M Tris-HCl (pH 9.1).

[0185] Alternatively, phagemid library panning was performed by KingFisher (Thermo Scientific) using Dynabeads (M280, streptavidin, Invitrogen, Cat. No. 60210) according to the manufacturer's instructions. 300 μL of Dynabeads were washed with PBS and incubated with biotinylated anti-human Fc for 20 min at room temperature. The beads were then blocked with 5% BSA in PBS for 1 h at room temperature. Fc fusion antigen (70-100 pmol) was captured by incubation for 1 h at room temperature. The beads were then washed once with PBS and incubated with 1 mL of phage library solution (typically 5*10 in 5% BSA-PBS). 12 ~1*10 13 The beads were incubated with 100 μL of 0.2 M glycine-HCl for 10 min at room temperature. The eluted phage were immediately neutralized with 18 μL of 1 M Tris-HCl (pH 9.1). A total of 3 or 4 rounds of panning were performed against each of the antigens, and a >10-fold excess of purified human Fc was included to reduce background binding.

[0186] For some of the antigens tested, 2 mL of antigen (10-30 μg / mL) was used to coat the immunotubes overnight at 4° C. Volumes of blocking, washing, and elution solutions were increased as necessary.

[0187] Amplification of enriched phages The eluted enriched phage pool was further amplified as follows: ER2738 cells were infected with the eluted phagemid particles for 30 minutes at 37°C. The infected cells were then plated on 2xYT agar plates containing 2% glucose, 100 μg / mL ampicillin and 12.5 μg / mL tetracycline. Colonies were picked from the plates, grown in 100 ml of 2% glucose, 100 μg / mL ampicillin and 12.5 μg / mL tetracycline, and infected with M13KO7 helper phage. The amplified phage was purified and quantified by the process described above. Usually, phage eluted after the final round of panning was used to infect ER2738 cells, and the resulting ER2738 colonies were picked for supernatant ELISA screening assays.

[0188] Supernatant sandwich Elisa assay A sensitive sandwich Elisa assay was constructed to measure the Fab present in the bacterial supernatant. Microplates were coated with polyclonal anti-human IgG (Fab specific) (Sigma I5260) to capture the Fab present in the bacterial supernatant, and then HRP-labeled goat anti-human Fc was used to detect the amount of captured Fab. 450 was measured to determine Fab binding activity. Primary hits were defined as those with an ELISA signal at least twice background and are further characterized in the following example (Example 4).

[0189] The constructed library was used to screen 12 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) and two corresponding mouse targets (TAGT-8M and TAGT-10M). A total of 690 high affinity unique positive hits were identified using these 14 antigens. The majority of the variant groups (Table 2) were able to form antibodies that bound to different target antigens or cross-reacted between the two species (e.g., binding to TAGT-8H and TAGT-8M). The variant groups of confirmed binders were a subset of the designed variant groups shown in Table 2. The majority of the designed variants were also found in the confirmed binders (Table 3). (See design formulas in Table 2 versus the positive hit formulas in Table 3). Table 3: Formulas of positive hit HVR-H1 and HVR-H2 designed variant sequences TIFF2024178314000009.tif244170TIFF2024178314000010.tif33170

[0190] Example 4: In vitro antibody characterization Fabs corresponding to the primary hits identified in Example 3 above were tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in E. coli, and purified by Ni-NTA resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Affinity was measured by a ForteBio Octet RED96 System. Briefly, AHC sensors (anti-human IgG-Fc capture dip and read biosensors) were used to capture antigen Fc-His fusion proteins (Sino Biological #10039-H03H) immersed in wells containing purified Fabs diluted to 5-10 μg / mL in kinetics buffer (SForteBio, Anti-human IgG Capture (AHC) Biosensors, Product Insert 41-0072-PD (2008); see also Yang et al. (2016), Anal. Biochem. 508:78-96). The acquired ForteBio data were processed with Data Acquisition software 7.1, and the kinetic data were fitted to a 1:1 Langmuir binding model. The melting temperatures of the Fabs were measured by Differential Scanning Fluorimetry (DSF) assay. Briefly, temperature and fluorescence monitoring were performed using a qPCR instrument (real-time PCR). SYPRO® Orange was diluted from a 5000x stock solution 50x to 100x in PBS buffer, and 16 μl of each Fab (approximately 0.5 mg / ml) was added to each well of a 96-well microplate and mixed with 4 μl of 100x SYPRO® Orange. Fluorescence intensity was measured using a LightCycler® 480 System. 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 1.2-1.3°C per minute, and an equilibration time of 15 seconds was applied at each measurement temperature. Data was analyzed using the LightCycler® 480 Software. The midpoint of hydrophobic exposure, Tm, was defined as the temperature corresponding to the maximum of the first derivative of the first order fluorescence transition.(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 See also Mol.Biol.94:10.28.1-10.28.15).

[0191] 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 with less than 26% sequence identity. The sequence identity between the human antigen TAGT-8H and the mouse antigen TAGT-8M was 70%, whereas the sequence identity between the human antigen TAGT-10H and the mouse antigen TAGT-10M was 60%. Multiple antibodies with high affinity targeting 14 different antigens were successfully identified and selected from the dynamic library. The affinity of most binders was in the nanomolar range, but some even reached the subnanomolar range (Figure 2A). In addition, the identified binders showed good stability, the range of which is shown in Figure 2B.

[0192] Example 5: Application of the Dynamic Heavy Chain Library To further explore the robustness and flexibility of the heavy chain library, 7 Libraries were screened against the 14 target antigens described in Example 4 above by pairing heavy chains with various light chain libraries with diversities ranging from ∼280 and even a single light chain (i.e., a common light chain). The limits of diversity design in both heavy and light chain libraries were set by the physical size of each pairing partner (e.g., 10 7Dynamic light chain libraries with diversities ranging from ~280, 20, and even a single light chain were explored by tailoring the dynamic light chain libraries. The ability of these dynamic light chain libraries in pairing with the dynamic heavy chain libraries provided a strong rationale for library design in generating and engineering diverse antibody hits / leads against known challenging target antigens. Positive hits with high affinity were identified from each of the tested libraries, and a total of 690 unique positive hits were measured and confirmed by affinity data (Table 4). The ability to bind to different targets and their epitope mutations (including but not limited to subtle differences in epitope recognition between two species as shown by species cross-reactivity with human and mouse targets with approximately 60% sequence identity) were investigated. Positive hits were observed using combinations of HVR-H1_1, HVR-H1_2, or HVRH-1_3 with HVR-H2_1, HVR-H2_2, or HVRH-2_3, HVR-H2_4, HVR-H_5, HVRH-2_6, or HVR-H2_7. These results demonstrate the power and potential of using these dynamic hypervariable region units for the generation of antibody and protein libraries that, when grafted or engineered into antibody (and / or alternative protein) scaffolds, recognize a wide range of targets for therapeutic, diagnostic, and / or research reagents. The broad diversity (e.g., 10 7 The dynamic properties of these heavy chain hypervariable region units when paired with light chain libraries containing multiple sequences (ranging from ~280 to a single unique sequence) are a powerful demonstration of the dynamic antibody design concept to generate novel binding reagents useful in therapeutic, diagnostic and / or research settings. Table 4: Affinity data of confirmed hits TIFF2024178314000011.tif253170TIFF2024178314000012.tif255170TIFF2024178314000013.tif255170TIFF2024178314000014.tif255170TIFF2024178314000015.tif255170TIFF2024178314000016.tif255170TIFF2024178314000017.tif255170TIFF2024178314000018.tif255170TIFF2024178314000019.tif255170TIFF2024178314000020.tif255170TIFF2024178314000021.tif255170TIFF2024178314000022.tif255170TIFF2024178314000023.tif255170TIFF2024178314000024.tif255170TIFF2024178314000025.tif255170TIFF2024178314000026.tif255170TIFF2024178314000027.tif255170TIFF2024178314000028.tif255170TIFF2024178314000029.tif255170TIFF2024178314000030.tif255170TIFF2024178314000031.tif255170TIFF2024178314000032.tif255170TIFF2024178314000033.tif255170TIFF2024178314000034.tif255170TIFF2024178314000035.tif255170TIFF2024178314000036.tif255170TIFF2024178314000037.tif255170TIFF2024178314000038.tif255170TIFF2024178314000039.tif255170TIFF2024178314000040.tif255170TIFF2024178314000041.tif255170TIFF2024178314000042.tif255170TIFF2024178314000043.tif255170TIFF2024178314000044.tif255170TIFF2024178314000045.tif79170.

[0193] It was discovered that hits containing the same HVR-H1 and HVR-H2 sequences can bind to different target antigens when these HVR-H1 and H2 sequences are paired with different HVR-H3 and VL sequences. For example, hit IDs 4029, 7097, and 5906 contain the same combination of HVR-H1 and HVR-H2 (HVR-H1_2 and HVR-H2_4), but when paired with different HVR-H3 and VL sequences, they bound to three different target antigens (TAGT-8, TAGT-6, and TAGT-12, respectively). Hits 7040 and 5924 contain the same combination of HVR-H1 and HVR-H2 (HVR-H1_2 and HVR-H2_6), but when paired with different HVR-H3 and VL sequences, they bound to two different target antigens (TAGT-8 and TAGT-12, respectively).

[0194] Table 5 below shows the sequence utilization and target binding numbers for HVR-H1 and HVR-H2 identified during library analysis. Without wishing to be bound by theory, it is believed that a high number of antigens bound by an antibody containing a given hypervariable region may indicate a high degree of flexibility for that particular hypervariable region, whereas a high segment utilization of a given hypervariable region may indicate robust folding of the hypervariable region (and surrounding polypeptide sequence). Table 5: Target binding capabilities of HVR-H1 and HVR-H2 designed variants TIFF2024178314000046.tif148170

[0195] Table 6 below shows the sequence utilization and antigen binding numbers of HVR-H1 and HVR-H2 combinations identified during library analysis. Table 6: Combination usage of HVR-H1 and HVR-H2 design variants TIFF2024178314000047.tif249170TIFF2024178314000048.tif41170

[0196] 74 HVR-H1 sequences (SEQ ID NOs: 1-52 and 137-158, Table 1) and 90 HVR-H2 sequences (SEQ ID NOs: 53-136 and 159-164, Table 1) were identified that appeared in more than one of the unique antibody hits above. These HVRs, when combined with various HVR-H3 and variable light domains, were able to form antibodies that bound to multiple antigens. An additional 65 new HVR-H1 and HVR-H2 sequence combinations were identified that appeared in more than one of the unique antibody hits above. Table 7 below shows the HVR-H1 and HVR-H2 usage and antigen binding numbers when libraries were analyzed using these new HVR sequences. Table 7: New HVR-H1 and HVR-H2 sequence usage TIFF2024178314000049.tif149170TIFF2024178314000050.tif251170TIFF2024178314000051.tif252170TIFF2024178314000052.tif245170

[0197] Table 8 below shows the combined use and antigen binding numbers of the new HVR-H1 and HVR-H2 sequences. Table 8: Combination use of the new HVR-H1 and HVR-H2 TIFF2024178314000053.tif227170TIFF2024178314000054.tif208170

[0198] Table 9 shows affinity data for the unique hits using the new HVR-H1 and HVR-H2 sequences shown. Table 9: Affinity data of confirmed hits using new HVR-H1 and HVR-H2 sequences TIFF2024178314000055.tif248170TIFF2024178314000056.tif255170TIFF2024178314000057.tif254170TIFF2024178314000058.tif255170TIFF2024178314000059.tif255170TIFF2024178314000060.tif255170TIFF2024178314000061.tif255170TIFF2024178314000062.tif255170TIFF2024178314000063.tif255170TIFF2024178314000064.tif255170TIFF2024178314000065.tif255170TIFF2024178314000066.tif255170TIFF2024178314000067.tif255170TIFF2024178314000068.tif255170TIFF2024178314000069.tif255170TIFF2024178314000070.tif255170TIFF2024178314000071.tif255170TIFF2024178314000072.tif255170TIFF2024178314000073.tif255170TIFF2024178314000074.tif255170TIFF2024178314000075.tif255170TIFF2024178314000076.tif255170TIFF2024178314000077.tif255170TIFF2024178314000078.tif255170TIFF2024178314000079.tif255170TIFF2024178314000080.tif255170TIFF2024178314000081.tif255170TIFF2024178314000082.tif255170TIFF2024178314000083.tif255170TIFF2024178314000084.tif255170TIFF2024178314000085.tif255170TIFF2024178314000086.tif255170TIFF2024178314000087.tif255170TIFF2024178314000088.tif255170TIFF2024178314000089.tif255170TIFF2024178314000090.tif25 5170TIFF2024178314000091.tif255170TIFF2024178314000092.tif255170TIFF2024178314000093.tif255170T IFF2024178314000094.tif255170TIFF2024178314000095.tif255170TIFF2024178314000096.tif255170TIFF20 24178314000097.tif255170TIFF2024178314000098.tif255170TIFF2024178314000099.tif255170TIFF2024178 314000100.tif255170TIFF2024178314000101.tif255170TIFF2024178314000102.tif255170TIFF202417831400 0103.tif255170TIFF2024178314000104.tif255170TIFF2024178314000105.tif255170TIFF2024178314000106. tif255170TIFF2024178314000107.tif255170TIFF2024178314000108.tif255170TIFF2024178314000109.tif25 5170TIFF2024178314000110.tif255170TIFF2024178314000111.tif255170TIFF2024178314000112.tif130170.

[0199] HVR-H1 comprising SEQ ID NO: 16 was used in 8 unique hits. With the same HVR-H1 sequence but different sequences for the other HVRs, these 8 hits had the ability to bind to 5 different target antigens. Exemplary hit IDs 4034, 6010, and 7183 bind to TAGT-8, TAGT-12, and TAGT-6, respectively, and contained HVR-H1 comprising SEQ ID NO: 16.

[0200] HVR-H2 containing SEQ ID NO:63 was used in 40 unique hits. With the same HVR-H2 sequence but different sequences for the other HVRs, these 40 hits had the ability to bind to seven different target antigens. Exemplary hit IDs 4036, 5115, and 5404 bind to TAGT-8, TAGT-12, and TAGT-6, respectively, and contained HVR-H2 comprising SEQ ID NO:63.

[0201] Exemplary hit IDs 3757 and 5103 contain the same HVR-H1 and HVR-H2 sequences (SEQ ID NOs: 1 and 122) and contain the same heavy chain variable region, but when combined with different variable light chain domains, bound to two different target antigens (TAGT-6 and TAGT-10, respectively). Two additional hits with these same HVR-H1 and HVR-H2 sequences were able to bind to another target antigen, TAGT-11.

[0202] Exemplary hit ID 4027, containing the HVR-H1 and HVR-H2 sequences of SEQ ID NOs: 31 and 124, was able to bind to the same antigen (TAGT-8H and TAGT-8M) from two different species. Some of the other hits with these same HVR-H1 and HVR-H2 sequences showed species cross-reactivity.

[0203] A novel method employing redefined antibody hypervariable regions based on structural and sequence variability to identify dynamic motifs has been developed. H The components are paired together in V L A limited number of V that can bind to the same or multiple different targets depending on the segment H The data and antibodies described herein provide a valuable tool in antibody discovery, whether the heavy chain library is used in its entirety or as a subset. H They show that it is robust enough to function as a building block. array Unless otherwise indicated, all polypeptide sequences are written from N-terminus to C-terminus. Unless otherwise indicated, all polynucleotide sequences are written 5' to 3'. Designed HVR-H1 sequence 1: FTFTDYGIHWV (SEQ ID NO: 1) Designed HVR-H1 sequence 2: FFTTGYAIHWV (SEQ ID NO: 2) Designed HVR-H1 sequence 3: FTFTNYGIHWV (SEQ ID NO: 3) Designed HVR-H1 sequence 4: YTFSDYAIHWV (SEQ ID NO: 4) Designed HVR-H1 sequence 5: YTFSDYGIHWV (SEQ ID NO: 5) Designed HVR-H1 sequence 6: YTFSGYAIHWV (SEQ ID NO: 6) Designed HVR-H1 sequence 7: YTFSGYGIHWV (SEQ ID NO: 7) Designed HVR-H1 sequence 8: YTFSNYGIHWV (SEQ ID NO: 8) Designed HVR-H1 sequence 9: YTFSSYGIHWV (SEQ ID NO: 9) Designed HVR-H1 sequence 10: YTFSGYWIHWV (SEQ ID NO: 10) Designed HVR-H1 sequence 11: YTFSNYWIHWV (SEQ ID NO: 11) Designed HVR-H1 sequence 12: FTFSGYWIHWV (SEQ ID NO: 12) Designed HVR-H1 sequence 13: FTFSNYWIHWV (SEQ ID NO: 13) Designed HVR-H1 sequence 14: YTFSDYWIHWV (SEQ ID NO: 14) Designed HVR-H1 sequence 15: YSISSGHHWAWI (SEQ ID NO: 15) Designed HVR-H1 sequence 16: YSISSGHYWNWI (SEQ ID NO: 16) Designed HVR-H1 sequence 17: YSISSGHYWSWI (SEQ ID NO: 17) Designed HVR-H1 sequence 18: YSISSGHYWTWI (SEQ ID NO: 18) Designed HVR-H1 sequence 19: YSISSGYHWAWI (SEQ ID NO: 19) Designed HVR-H1 sequence 20: YSISSGYHWDWI (SEQ ID NO: 20) Designed HVR-H1 sequence 21: YSISSGYHWGWI (SEQ ID NO: 21) Designed HVR-H1 sequence 22: YSISSGYHWNWI (SEQ ID NO: 22) Designed HVR-H1 sequence 23: YSISSGYHWSWI (SEQ ID NO: 23) Designed HVR-H1 sequence 24: YSISSGHHWDWI (SEQ ID NO: 24) Designed HVR-H1 sequence 25: YSISSGYYWDWI (SEQ ID NO: 25) Designed HVR-H1 sequence 26: YSISSGYYWNWI (SEQ ID NO: 26) Designed HVR-H1 sequence 27: YSISSGYYWTWI (SEQ ID NO: 27) Designed HVR-H1 sequence 28: YSITSGHHWAWI (SEQ ID NO: 28) Designed HVR-H1 sequence 29: YSITSGHHWDWI (SEQ ID NO: 29) Designed HVR-H1 sequence 30: YSITSGHHWGWI (SEQ ID NO: 30) Designed HVR-H1 sequence 31: YSITSGHHWNWI (SEQ ID NO:31) Designed HVR-H1 sequence 32: YSITSGHHWSWI (SEQ ID NO: 32) Designed HVR-H1 sequence 33: YSISSGHHWGWI (SEQ ID NO: 33) Designed HVR-H1 sequence 34: YSITSGHYWAWI (SEQ ID NO:34) Designed HVR-H1 sequence 35: YSITSGHYWDWI (SEQ ID NO: 35) Designed HVR-H1 sequence 36: YSITSGHYWGWI (SEQ ID NO: 36) Designed HVR-H1 sequence 37: YSITSGHYWNWI (SEQ ID NO:37) Designed HVR-H1 sequence 38: YSITSGHYWSWI (SEQ ID NO: 38) Designed HVR-H1 sequence 39: YSITSGYHWAWI (SEQ ID NO: 39) Designed HVR-H1 sequence 40: YSITSGYHWGWI (SEQ ID NO: 40) Designed HVR-H1 sequence 41: YSISSGHHWNWI (SEQ ID NO: 41) Designed HVR-H1 sequence 42: YSITSGYHWNWI (SEQ ID NO: 42) Designed HVR-H1 sequence 43: YSITSGYHWSWI (SEQ ID NO: 43) Designed HVR-H1 sequence 44: YSITSGYYWDWI (SEQ ID NO: 44) Designed HVR-H1 sequence 45: YSISSGHHWTWI (SEQ ID NO: 45) Designed HVR-H1 sequence 46: YSISSGHYWDWI (SEQ ID NO: 46) Designed HVR-H1 sequence 47: FSLSTSGVAVSWI (SEQ ID NO: 47) Designed HVR-H1 sequence 48: FSLSTGGVAVGWI (SEQ ID NO: 48) Designed HVR-H1 sequence 49: FSLSTGGVAVSWI (SEQ ID NO: 49) Designed HVR-H1 sequence 50: FSLSTGGVGVAWI (SEQ ID NO: 50) Designed HVR-H1 sequence 51: FSLSTGGVGVSWI (SEQ ID NO:51) Designed HVR-H1 sequence 52: FSLSTSGVAVAWI (SEQ ID NO:52) Designed HVR-H1 sequence 53: FTFSDYAIHWV (SEQ ID NO: 137) Designed HVR-H1 sequence 54: FTFSDYGIHWV (SEQ ID NO: 138) Designed HVR-H1 sequence 55: YTFSNYAIHWV (SEQ ID NO: 139) Designed HVR-H1 sequence 56: YTFSSYAIHWV (SEQ ID NO: 140) Designed HVR-H1 sequence 57: YTFTDYAIHWV (SEQ ID NO: 141) Designed HVR-H1 sequence 58: YTFTDYGIHWV (SEQ ID NO: 142) Designed HVR-H1 sequence 59: YTFTNYAIHWV (SEQ ID NO: 143) Designed HVR-H1 sequence 60: YTFTNYGIHWV (SEQ ID NO: 144) Designed HVR-H1 sequence 61: FTFSGYGIHWV (SEQ ID NO: 145) Designed HVR-H1 sequence 62: FTFSNYAIHWV (SEQ ID NO: 146) Designed HVR-H1 sequence 63: FTFSSYGIHWV (SEQ ID NO: 147) Designed HVR-H1 sequence 64: FTFSDYWIHWV (SEQ ID NO: 148) Designed HVR-H1 sequence 65: FTFTSYWIHWV (SEQ ID NO: 149) Designed HVR-H1 sequence 66: YSISSGYYWGWI (SEQ ID NO: 150) Designed HVR-H1 sequence 67: YSITSGYYWNWI (SEQ ID NO: 151) Designed HVR-H1 sequence 68: YSITSGYYWSWI (SEQ ID NO: 152) Designed HVR-H1 sequence 69: YSISSGHYWAWI (SEQ ID NO: 153) Designed HVR-H1 sequence 70: YSISSGHYWGWI (SEQ ID NO: 154) Designed HVR-H1 sequence 71: FSLSTSGVAVGWI (SEQ ID NO: 155) Designed HVR-H1 sequence 72: FSLSTSGVGVAWI (SEQ ID NO: 156) Designed HVR-H1 sequence 73: FSLSTSGVGVGWI (SEQ ID NO: 157) Designed HVR-H1 sequence 74: FSLSTGGVGVGWI (SEQ ID NO: 158) Designed HVR-H2 sequence 1: LARIDWDDDKRYSPSLKSRL (SEQ ID NO: 53) Designed HVR-H2 sequence 2: LALIDWDDDKRYSPSLKSRL (SEQ ID NO:54) Designed HVR-H2 sequence 3: LALIDWDDDKRYSTSLKSRL (SEQ ID NO:55) Designed HVR-H2 sequence 4: LALIDWDDDKYYSPSLKSRL (SEQ ID NO:56) Designed HVR-H2 sequence 5: LALIDWADDKYYSPSLKSRL (SEQ ID NO:57) Designed HVR-H2 sequence 6: LALIDWAGDKSYSTSLKSRL (SEQ ID NO:58) Designed HVR-H2 sequence 7: LARIDWDDDKYYSPSLKSRL (SEQ ID NO:59) Designed HVR-H2 sequence 8: LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 60) Designed HVR-H2 sequence 9: LARIDWDGDKYYSTSLKSRL (SEQ ID NO: 61) Designed HVR-H2 sequence 10: IGDIYHSGSTYYSPSLKSRV (SEQ ID NO: 62) Designed HVR-H2 sequence 11: IGEIYHSGSTYYSPSLKSRV (SEQ ID NO: 63) Designed HVR-H2 sequence 12: IGEIYYSGSTYYSPSLKSRV (SEQ ID NO: 64) Designed HVR-H2 sequence 13: IGSIYHSGNTNYNPSLKSRV (SEQ ID NO: 65) Designed HVR-H2 sequence 14: IGEIYHSGNTYYNPSLKSRV (SEQ ID NO: 66) Designed HVR-H2 sequence 15: IGEIYHSGSTYYNPSLKSRV (SEQ ID NO: 67) Designed HVR-H2 sequence 16: IGEIYYSGSTYYNPSLKSRV (SEQ ID NO: 68) Designed HVR-H2 sequence 17: IGDIYHSGNTYYNPSLKSRV (SEQ ID NO: 69) Designed HVR-H2 sequence 18: IGDIYHSGSTYYNPSLKSRV (SEQ ID NO: 70) Designed HVR-H2 sequence 19: VSAISGYGDTTYYADSVKGRF (SEQ ID NO: 71) Designed HVR-H2 sequence 20: VSAISGYGGSTYYADSVKGRF (SEQ ID NO: 72) Designed HVR-H2 sequence 21: VSAISGYGGTTYYADSVKGRF (SEQ ID NO: 73) Designed HVR-H2 sequence 22: VSGISGAGDTTYYADSVKGRF (SEQ ID NO: 74) Designed HVR-H2 sequence 23: VSGISGDGDTTYYADSVKGRF (SEQ ID NO: 75) Designed HVR-H2 sequence 24: VSGISGDGGSTYYADSVKGRF (SEQ ID NO: 76) Designed HVR-H2 sequence 25: VSGISGYGDTTYYADSVKGRF (SEQ ID NO: 77) Designed HVR-H2 sequence 26: VSGISGYGGTTYYADSVKGRF (SEQ ID NO: 78) Designed HVR-H2 sequence 27: VSVISGDGDTTYYADSVKGRF (SEQ ID NO: 79) Designed HVR-H2 sequence 28: VSVISGYGGSTYYADSVKGRF (SEQ ID NO: 80) Designed HVR-H2 sequence 29: VSGISGDGSTTYYADSVKGRF (SEQ ID NO: 81) Designed HVR-H2 sequence 30: VSGISGYGSTTYYADSVKGRF (SEQ ID NO: 82) Designed HVR-H2 sequence 31: VSVISGSGSTTYADSVKGRF (SEQ ID NO: 83) Designed HVR-H2 sequence 32: VSVISGYGSSTYYADSVKGRF (SEQ ID NO: 84) Designed HVR-H2 sequence 33: VSVISGYGSTTYYADSVKGRF (SEQ ID NO: 85) Designed HVR-H2 sequence 34: VSAISGYGSTTYYADSVKGRF (SEQ ID NO: 86) Designed HVR-H2 sequence 35: VSSISGYGDTTYYADSVKGRF (SEQ ID NO: 87) Designed HVR-H2 sequence 36: VSSISGYGGSTYYADSVKGRF (SEQ ID NO: 88) Designed HVR-H2 sequence 37: VSSISGYGGTTYYADSVKGRF (SEQ ID NO: 89) Designed HVR-H2 sequence 38: VSYISGAGDTTYYADSVKGRF (SEQ ID NO: 90) Designed HVR-H2 sequence 39: VSSISGAGDTTYYADSVKGRF (SEQ ID NO: 91) Designed HVR-H2 sequence 40: VSYISGAGGTTYYADSVKGRF (SEQ ID NO: 92) Designed HVR-H2 sequence 41: VSYISGDGDTTYYADSVKGRF (SEQ ID NO: 93) Designed HVR-H2 sequence 42: VSYISGDGGSTYYADSVKGRF (SEQ ID NO: 94) Designed HVR-H2 sequence 43: VSYISGDGGTTYYADSVKGRF (SEQ ID NO: 95) Designed HVR-H2 sequence 44: VSYISGSGDTTYYADSVKGRF (SEQ ID NO: 96) Designed HVR-H2 sequence 45: VSSISGAGGSTYYADSVKGRF (SEQ ID NO: 97) Designed HVR-H2 sequence 46: VSYISGYGDTTYYADSVKGRF (SEQ ID NO: 98) Designed HVR-H2 sequence 47: VSYISGYGGTTYYADSVKGRF (SEQ ID NO: 99) Designed HVR-H2 sequence 48: VSSISGAGGTTYYADSVKGRF (SEQ ID NO: 100) Designed HVR-H2 sequence 49: VSSISGDGDTTYYADSVKGRF (SEQ ID NO: 101) Designed HVR-H2 sequence 50: VSSISGDGGTTYYADSVKGRF (SEQ ID NO: 102) Designed HVR-H2 sequence 51: VSSISGAGSSTYYADSVKGRF (SEQ ID NO: 103) Designed HVR-H2 sequence 52: VSSISGAGSTTYYADSVKGRF (SEQ ID NO: 104) Designed HVR-H2 sequence 53: VSSISGDGSSTYYADSVKGRF (SEQ ID NO: 105) Designed HVR-H2 sequence 54: VSSISGDGSTTYYADSVKGRF (SEQ ID NO: 106) Designed HVR-H2 sequence 55: VSSISGYGSSTYYADSVKGRF (SEQ ID NO: 107) Designed HVR-H2 sequence 56: VSSISGYGSTTYYADSVKGRF (SEQ ID NO: 108) Designed HVR-H2 sequence 57: IGWINPNRGDTKYAQKFQGRV (SEQ ID NO: 109) Designed HVR-H2 sequence 58: IGWINPNRGDTNYAQKFQGRV (SEQ ID NO: 110) Designed HVR-H2 sequence 59: IGWINPNRGGTKYAQKFQGRV (SEQ ID NO: 111) Designed HVR-H2 sequence 60: IGWINPNRGGTNYAQKFQGRV (SEQ ID NO: 112) Designed HVR-H2 sequence 61: IGWINPNRGSTKYAQKFQGRV (SEQ ID NO: 113) Designed HVR-H2 sequence 62: IGWINPNRGSTNYAQKFQGRV (SEQ ID NO: 114) Designed HVR-H2 sequence 63: IGRINPNNFGDTNYAQKFQGRV (SEQ ID NO: 115) Designed HVR-H2 sequence 64: IGWINPNFGDTNYAQKFQGRV (SEQ ID NO: 116) Designed HVR-H2 sequence 65: IGWINPNFGSTKYAQKFQGRV (SEQ ID NO: 117) Designed HVR-H2 sequence 66: IGWINPNFGSTNYAQKFQGRV (SEQ ID NO: 118) Designed HVR-H2 sequence 67: IGIINPNRGDTKYAQKFQGRV (SEQ ID NO: 119) Designed HVR-H2 sequence 68: IGIINPNRGDTNYAQKFQGRV (SEQ ID NO: 120) Designed HVR-H2 sequence 69: IGIINPNFGDTNYAQKFQGRV (SEQ ID NO: 121) Designed HVR-H2 sequence 70: IGWISPSGGGTKYAQKFQGRV (SEQ ID NO: 122) Designed HVR-H2 sequence 71: IGWISPSGGGTNYAQKFQGRV (SEQ ID NO: 123) Designed HVR-H2 sequence 72: IGWISPSSGGTKYAQKFQGRV (SEQ ID NO: 124) Designed HVR-H2 sequence 73: IGWISPSSGGTNYAQKFQGRV (SEQ ID NO: 125) Designed HVR-H2 sequence 74: IGWIYPSGGGTKYAQKFQGRV (SEQ ID NO: 126) Designed HVR-H2 sequence 75: IGWIYPSGGGTNYAQKFQGRV (SEQ ID NO: 127) Designed HVR-H2 sequence 76: IGWISPSGGSTNYAQKFQGRV (SEQ ID NO: 128) Designed HVR-H2 sequence 77: IGWISPSSGSTKYAQKFQGRV (SEQ ID NO: 129) Designed HVR-H2 sequence 78: IGWISPSSGSTNYAQKFQGRV (SEQ ID NO: 130) Designed HVR-H2 sequence 79: IGWISPSGGSTKYAQKFQGRV (SEQ ID NO: 131) Designed HVR-H2 sequence 80: IGIIYPSGGGTNYAQKFQGRV (SEQ ID NO: 132) Designed HVR-H2 sequence 81: IGIISPSGGGTKYAQKFQGRV (SEQ ID NO: 133) Designed HVR-H2 sequence 82: IGIISPSGGGTNYAQKFQGRV (SEQ ID NO: 134) Designed HVR-H2 sequence 83: IGIIYPSGGSTNYAQKFQGRV (SEQ ID NO: 135) Designed HVR-H2 sequence 84: VGRIKSKTDGYTTEYAAPVKGRF (SEQ ID NO: 136) Designed HVR-H2 sequence 85: VSAISGSGSTTYADSVKGRF (SEQ ID NO: 159) Designed HVR-H2 sequence 86: VSSISGSGDTTYYADSVKGRF (SEQ ID NO: 160) Designed HVR-H2 sequence 87: VSSISGSGGSTYYADSVKGRF (SEQ ID NO: 161) Designed HVR-H2 sequence 88: VSSISGSGGTTYYADSVKGRF (SEQ ID NO: 162) Designed HVR-H2 sequence 89: VSSISGDGGSTYYADSVKGRF (SEQ ID NO: 163) Designed HVR-H2 sequence 90: VSSISGSGSTTYYADSVKGRF (SEQ ID NO: 164) Framework FW-H1 sequence: EVQLVESGGGLVQPGGSLRLSCAASG (SEQ ID NO: 165) Framework FW-H2 sequence: RQAPGKGLEW (SEQ ID NO: 166) Framework FW-H3 sequence: TISSRDNSKNTLYLQLNSLRAEDTAVYYC (SEQ ID NO: 167) Framework FW-H4 sequence: WGQGTLVTVSS (SEQ ID NO: 168) Hit ID 4029-VH EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYHWGWIRQAPGKGLEWVSYISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDYGDYYGFDYWGQGTLVTVSS (SEQ ID NO: 169) Hit ID 4029-VL DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVEIKR (SEQ ID NO: 170) Hit ID 7097-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHHWDWIRQAPGKGLEWVSYISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGSDAVLGDWFAYWGQGTLVTVSS (SEQ ID NO: 171) Hit ID 7097-VL DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGTKVEIKR (SEQ ID NO: 172) Hit ID 5906-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWNWIRQAPGKGLEWVSYISGDGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLGGYYGWGRYFDYWGQGTLVTVSS (SEQ ID NO: 173) Hit ID 5906-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR (SEQ ID NO: 174) Hit ID 7040-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWNWIRQAPGKGLEWIGWISPSGGSTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLTAGGFDYWGQGTLVTVSS (SEQ ID NO: 175) Hit ID 7040-VL DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGTKVEIKR (SEQ ID NO: 176) Hit ID 5924-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWGWIRQAPGKGLEWIGIISPSSGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGAGVHYALDYWGQGTLVTVSS (SEQ ID NO: 177) Hit ID 5924-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR (SEQ ID NO: 178) Hit ID 4034-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWVSSISGYGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARERYYGSTDYAFDYWGQGTLVTVSS (SEQ ID NO: 179) Hit ID 4034-VL DIQLTQSPSSLSASVGDRVTITCSASSRVSHVFWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCLQGTHFPWTFGQGTKVEIKR (SEQ ID NO: 180) Hit ID 6010-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWIGWINPNRGDTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDYYGDFDYWGQGTLVTVSS (SEQ ID NO: 181) Hit ID 6010-VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (SEQ ID NO: 182) Hit ID 7183-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWVSSISGYGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGSDTVLGDWFAYWGQGTLVTVSS (SEQ ID NO: 183) Hit ID 7183-VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKR (SEQ ID NO: 184) Hit ID 4036-VH EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGVGVGWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARERYGSYYFDYWGQGTLVTVSS (SEQ ID NO: 185) Hit ID 4036-VL DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGKSFLDWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRIPPTFGQGTKVEIKR (SEQ ID NO: 186) Hit ID 5115-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWGWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARESYYAFDYWGQGTLVTVSS (SEQ ID NO: 187) Hit ID 5115-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTPLTFGQGTKVEIKR (SEQ ID NO: 188) Hit ID 5404-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWAWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISR DNSKNTLYLQLNSLRAEDTAVYYCARSPYYYGVFDYWGQGTLVTVSS (SEQ ID NO: 189) Hit ID 5404-VL DIQLTQSPSSLSASVGDRVTITCSASSRVGSVYWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYTHDPVTFGQGTKVEIKR (SEQ ID NO: 190) Hit ID 3757-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGVGDFDYWGQGTLVTVSS (SEQ ID NO: 191) Hit ID 3757-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR (SEQ ID NO: 192) Hit ID 5103-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGVGDFDYWGQGTLVTVSS (SEQ ID NO: 193) Hit ID 5103-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR (SEQ ID NO: 194) Hit ID 4027-VH EVQLVESGGGLVQPGGSLRLSCAASGYSITSGHHWNWIRQAPGKGLEWIGWISPSSGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGFDGFHYWGQGTLVTVSS (SEQ ID NO: 195) Hit ID 4027-VL DIQLTQSPSSLSASVGDRVTITCRASESVDFYGISFLPWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPWTFGQGTKVEIKR (SEQ ID NO: 196) VH in Figure 1B EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYGIHWVRQAPGKGLEWVSGISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARERDYDFDYWGQGTLVTVSS (SEQ ID NO: 197) 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) 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) 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) 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) 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) Formula (XIII) 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) Primer F_1999 CGTTTGTCCTGTGCAGCTTCCGG (SEQ ID NO: 211) Primer R_1999 CGAGGCCCTTACCCGGGGCCTGACG (SEQ ID NO: 212) Primer F_2003 CCGGGTAAGGGCCTCGAGTGG (SEQ ID NO: 213) Primer R_2003 GAGCACGTCCGTTCGAATTGTCGCGACTTATAG (SEQ ID NO: 214) Primer S1089 ACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTG (SEQ ID NO: 215) Primer S1090 GAGGAGACGGTGACTAGTGTTCCTTGACCCCA (SEQ ID NO: 216) Primer F_2898 TACTTATGTAGGCGATCGGGTCACCATCACCTGC (SEQ ID NO: 217) Primer R_2898 CGGAGCTTTTCCTGGTTTCTGTTGATAC (SEQ ID NO: 218) Primer F_2013 GAAACCAGGAAAAGCTCCGAAG (SEQ ID NO: 219) Primer R_2013 CGTCCCGGAACCGGATCCAGAGAAGCGAG (SEQ ID NO: 220) Primer F2929 ACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAAC (SEQ ID NO: 221) Primer R2929 GATCTCCACCTTGGTACCCTGTCCGAA (SEQ ID NO: 222) HVR-H3 sequence 1: ARDLGGYYGWGRYFDY (SEQ ID NO: 223) HVR-H3 sequence 2: ARDLTAGGFDY (SEQ ID NO: 224) HVR-H3 sequence 3: ARDPGVGGFDV (SEQ ID NO: 225) HVR-H3 sequence 4: ARDPGYTWYFDV (SEQ ID NO: 226) HVR-H3 sequence 5: ARDYGDYYGFDY (SEQ ID NO: 227) HVR-H3 sequence 6: ARDYGYTWYFDV (SEQ ID NO: 228) HVR-H3 sequence 7: ARDYYGDFDY (SEQ ID NO: 229) HVR-H3 sequence 8: AREGSDAVLGDWFAY (SEQ ID NO: 230) HVR-H3 sequence 9: AREGSDTVLGDWFAY (SEQ ID NO: 231) HVR-H3 sequence 10: ARERYGSYYFDY (SEQ ID NO: 232) HVR-H3 sequence 11: ARERYYGSTDYAFDY (SEQ ID NO: 233) HVR-H3 sequence 12: ARESYYAFDY (SEQ ID NO: 234) HVR-H3 sequence 13: ARGAGVHYALDY (SEQ ID NO: 235) HVR-H3 sequence 14: ARGFDGFHY (SEQ ID NO: 236) HVR-H3 sequence 15: ARGFYGGALDV (SEQ ID NO: 237) HVR-H3 sequence 16: ARGGGGYYFDV (SEQ ID NO: 238) HVR-H3 sequence 17: ARGGGLGFDY (SEQ ID NO: 239) HVR-H3 sequence 18: ARGGLGPFDI (SEQ ID NO: 240) HVR-H3 sequence 19: ARGGSDTVIGDWFAY (SEQ ID NO: 241) HVR-H3 sequence 20: ARGGVGPFDI (SEQ ID NO: 242) HVR-H3 sequence 21: ARGGYGGYLDV (SEQ ID NO: 243) HVR-H3 sequence 22: ARGLSSGYFDY (SEQ ID NO: 244) HVR-H3 sequence 23: ARGSWYFDV (SEQ ID NO: 245) HVR-H3 sequence 24: ARGTRGLDY (SEQ ID NO: 246) HVR-H3 sequence 25: ARGYSDYFDY (SEQ ID NO: 247) HVR-H3 sequence 26: ARGYYYGRAFDY (SEQ ID NO: 248) HVR-H3 sequence 27: ARHSYYGVGDFDY (SEQ ID NO: 249) HVR-H3 sequence 28: ARLFEGFPY (SEQ ID NO: 250) HVR-H3 sequence 29: ARLYDYFAY (SEQ ID NO: 251) HVR-H3 sequence 30: ARSGYYALDY (SEQ ID NO: 252) HVR-H3 sequence 31: ARSPYYYGVFDY (SEQ ID NO: 253) HVR-H3 sequence 32: ARSYVYFDY (SEQ ID NO: 254) HVR-H3 sequence 33: ARDGLGLRGVYYYYYGLDV (SEQ ID NO: 255) HVR-H3 sequence 34: ARVESGGIESPYYYYGLDV (SEQ ID NO: 256) HVR-L1 sequence 1: RASESVDFYGISFLP (SEQ ID NO: 257) HVR-L1 sequence 2: RASQSVDFYGISFLA (SEQ ID NO: 258) HVR-L1 sequence 3: RASQSVDFYGKSFLD (SEQ ID NO: 259) HVR-L1 sequence 4: SASSRVGSVY (SEQ ID NO: 260) HVR-L1 sequence 5: SASSRVSHVF (SEQ ID NO: 261) HVR-L1 sequence 6: RASQGISSYLA (SEQ ID NO: 262) HVR-L1 sequence 7: RASQSVSSYLA (SEQ ID NO: 263) HVR-L1 sequence 8: RASQSISSYLN (SEQ ID NO: 264) HVR-L3 sequence 1: FCLQGTHFPWT (SEQ ID NO: 265) HVR-L3 sequence 2: YCQQSYRTPFT (SEQ ID NO: 266) HVR-L3 sequence 3: YCQQSYSWPWT (SEQ ID NO: 267) HVR-L3 sequence 4: YCQQYTHDPVT (SEQ ID NO: 268) HVR-L3 sequence 5: YCQQYYRIPPT (SEQ ID NO: 269) HVR-L3 sequence 6: YCQHHYGTPLT (SEQ ID NO: 270) HVR-L3 sequence 7: YCQQSYSTPLT (SEQ ID NO: 271) HVR-L3 sequence 8: YCQQSYSTPPT (SEQ ID NO: 272) HVR-L3 sequence 9: YCQQYYSTPLT (SEQ ID NO: 273) HVR-L3 sequence 10: YCQQYYTTPLT (SEQ ID NO: 274)

Claims

1. A library comprising polynucleotide members, all of which encode an antibody heavy chain variable region composed of three hypervariable regions (HVRs) and four framework (FW) regions arranged in the following order: (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4); All polynucleotide members encode an antibody heavy chain variable region comprising HVR-H1 and HVR-H2 as follows: The HVR-H1 is (Formula I) X 1 TFX 2 X 3 YX 4 IHWV (SEQ ID NO: 198), where X 1 is F or Y, X 2 is S or T, X 3 is D, G, N, or S, and X 4 is A, G, or W; (Formula II) YSIX 1 SGX 2 X 3 WX 4 WI (SEQ ID NO: 199), where X 1 is S or T, X 2 is H or Y, X 3 is H or Y, and X 4 is A, D, G, N, S, or T; and (Formula III) FSLSTX 1 GVX 2 VX 3 WI (SEQ ID NO: 200) (wherein X 1 is G or S, X 2 is A or G, and X 3 is A, G, S, or T). comprising an amino acid sequence according to a formula selected from the group consisting of: The HVR-H2 is (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL (SEQ ID NO:201), where 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; (Formula V) IGX 1 IX 2 X 3 SGSTYYSPSLKSRV (SEQ ID NO:202), where X 1 is A, D, E, S, or Y, X 2 is S or Y, and X 3 is H or Y; (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV (SEQ ID NO:203), where X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y; (Formula VII) VSX 1 ISGX 2 GX 3 X 4 TYYADSVKGRF (SEQ ID NO: 204), where X 1 is A, G, S, V, or Y, X 2 is A, D, S, or Y, X 3 is D, G, or S, and X 4 is S or T; (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV (SEQ ID NO:205), where X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N; (Formula IX) IGX 1 IX 2 PSX 3 GX 4 TX 5 YAQKFQGRV (SEQ ID NO:206), where X 1 is I, R, or W, X 2 is S or Y, X 3 is G or S, X 4 is D, G, or S, and X 5 is K or N; and (Formula X) VGRIX 1 SKX 2 X 3 GX 4 TTX 5 YAAX 6 VKGRF (SEQ ID NO: 207) (wherein X 1 is K or R, X 2 is A or T, X 3 is D or Y, X 4 is G or Y, X 5 is D or E, and X 6 is P or S). comprising an amino acid sequence according to a formula selected from the group consisting of: the heavy chain variable region comprises FW-H1 comprising the amino acid sequence of SEQ ID NO: 165, FW-H2 comprising the amino acid sequence of SEQ ID NO: 166, FW-H3 comprising the amino acid sequence of SEQ ID NO: 167, and / or FW-H4 comprising the amino acid sequence of SEQ ID NO: 168; the library further comprises at least one polynucleotide encoding an antibody light chain variable region; Library.

2. The library of claim 1, wherein the polynucleotides comprise unique combinations of HVR-H1 and HVR-H2 sequences that are less than 6.5*10 4 .

3. a. The polynucleotide comprises a unique combination of HVR-H1 and HVR-H2 sequences that is less than 6700; or b. The library of claim 2, wherein the polynucleotides comprise no more than 6660 unique combinations of HVR-H1 and HVR-H2 sequences.

4. a. The heavy chain variable region comprises HVR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and / or The library according to any one of claims 1 to 3, wherein the heavy chain variable region comprises HVR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164.

5. HVR-H1 and HVR-H2 are HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IX), HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII), HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (VII), HVR-H1 comprising the amino acid sequence of formula (I) and HVR-H2 comprising the amino acid sequence of formula (IX), HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (IV), HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (V), HVR-H1 comprising the amino acid sequence of formula (II) and HVR-H2 comprising the amino acid sequence of formula (VII), and HVR-H2 comprising an amino acid sequence of formula (VI), HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (VII), HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (VII), HVR-H1 comprising an amino acid sequence of formula (II) and HVR-H2 comprising an amino acid sequence of formula (VIII), HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (V), HVR-H1 comprising an amino acid sequence of formula (III) and HVR-H2 comprising an amino acid sequence of formula (V), and HVR-H1 comprising an amino acid sequence of formula (I) and HVR-H2 comprising an amino acid sequence of formula (VIII), The library of claim 1, selected from the group consisting of:

6. HVR-H1 and HVR-H2 are HVR-H1 comprising the amino acid sequence of SEQ ID NO: 157 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 122; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 154 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 161; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 145 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 128, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 61, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63, HVR-H1 comprising the amino acid sequence of SEQ ID NO: 153 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 63, the sequence HVR-H1 comprising the amino acid sequence of SEQ ID NO: 155 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 67; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 156 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 100; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 51 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 123; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 139 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110 HVR-H1 comprising the amino acid sequence of SEQ ID NO:8 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:126; HVR-H1 comprising the amino acid sequence of SEQ ID NO:13 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:129; HVR-H1 comprising the amino acid sequence of SEQ ID NO:31 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:124; HVR-H1 comprising the amino acid sequence of SEQ ID NO:25 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:130; HVR-H1 comprising the amino acid sequence of SEQ ID NO:150 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:132;HVR-H1 comprising the amino acid sequence of SEQ ID NO: 158 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 162; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 82; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 149 and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 117; and HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7 and HVR-H2 comprising the amino acid sequence of SEQ ID NO:

134. The library of claim 1, selected from the group consisting of:

7. A library described in any one of claims 1 to 6, wherein the heavy chain variable region comprises HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.

8. The library described in 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.

9. The library of claim 1, wherein the polynucleotide encodes a full-length antibody heavy chain.

10. a. the antibody light chain variable region comprises HVR-L1, HVR-L2 and 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; and / or b. the polynucleotide encoding the antibody light chain variable region comprises at least 1 unique sequence, at least 280 unique sequences, or at least 10 5 unique sequences; The library according to any one of claims 1 to 9.

11. At least one of the HVR-H1 and HVR-H2 of the antibody heavy chain variable region adopts a plurality of three-dimensional structures as assessed by structure determination and / or computer modeling. The library according to any one of claims 1 to 10.

12. A library described in any one of claims 1 to 11, wherein at least one of the polynucleotides encoding the antibody heavy chain variable region is in a vector.

13. The library described in claim 12, wherein the vector is an expression vector or a display vector.

14. A library described in any one of claims 1 to 13, wherein at least one of the polynucleotides encoding an antibody heavy chain variable region is present in a cell.

15. The library of claim 14, wherein the cells are bacterial, yeast, or mammalian cells.

16. A library described in any one of claims 1 to 15, comprising polynucleotides encoding multiple unique antibodies.

17. The library of claim 16, wherein the heavy chain variable regions of each of the multiple antibodies comprise an identical sequence.

18. A kit comprising a library described in any one of claims 1 to 17.

19. A method for preparing a library comprising providing and assembling polynucleotide sequences of the library described in any one of claims 1 to 17.

20. A non-human animal comprising a library described in any one of claims 1 to 17.

21. The non-human animal described in claim 20, wherein the non-human animal is a mammal.

22. A library comprising antigen-binding domains, wherein one of the antigen-binding domains comprises an antibody heavy chain variable region and an antibody light chain variable region defined in a library described in any one of claims 1 to 17.

23. The library of claim 22, wherein the library comprises phages and the antigen-binding domain is displayed on the surface of at least one phage in the library.

24. A method for producing a bispecific antibody comprising two identical antibody heavy chain variable regions and two identical antibody light chain variable regions, comprising: a. screening a library of first antigen-binding domains for binding to a first antigen, said library of first antigen-binding domains comprising antigen-binding domain members; All of the antigen-binding domain members comprise a first antibody heavy chain variable region and a first antibody light chain variable region, and all of the first antibody heavy chain variable regions are composed of three hypervariable regions (HVRs) and four framework (FW) regions arranged in the following order: (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4), and all of the first antibody heavy chain variable regions comprise the following HVR-H1 and HVR-H2: HVR-H1, (Formula I) X 1 TFX 2 X 3 YX 4 IHWV (SEQ ID NO: 198), where X 1 is F or Y, X 2 is S or T, X 3 is D, G, N, or S, and X 4 is A, G, or W; (Formula II) YSIX 1 SGX 2 X 3 WX 4 WI (SEQ ID NO: 199), where X 1 is S or T, X 2 is H or Y, X 3 is H or Y, and X 4 is A, D, G, N, S, or T; and (Formula III) FSLSTX 1 GVX 2 VX 3 WI (SEQ ID NO: 200) (wherein X 1 is G or S, X 2 is A or G, and X 3 is A, G, S, or T). comprising an amino acid sequence according to a formula selected from the group consisting of: HVR-H2, (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL (SEQ ID NO:201), where 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; (Formula V) IGX 1 IX 2 X 3 SGSTYYSPSLKSRV (SEQ ID NO:202), where X 1 is A, D, E, S, or Y, X 2 is S or Y, and X 3 is H or Y; (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV (SEQ ID NO:203), where X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y; (Formula VII) VSX 1 ISGX 2 GX 3 X 4 TYYADSVKGRF (SEQ ID NO: 204), where X 1 is A, G, S, V, or Y, X 2 is A, D, S, or Y, X 3 is D, G, or S, and X 4 is S or T; (Formula VIII) IGX1INPNX2GX3TX4YAQKFQGRV (SEQ ID NO:205), where X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N; (Formula IX) IGX 1 IX 2 PSX 3 GX 4 TX 5 YAQKFQGRV (SEQ ID NO:206), where X 1 is I, R, or W, X 2 is S or Y, X 3 is G or S, X 4 is D, G, or S, and X 5 is K or N; and (Formula X) VGRIX 1 SKX 2 X 3 GX 4 TTX 5 YAAX 6 VKGRF (SEQ ID NO: 207) (wherein X 1 is K or R, X 2 is A or T, X 3 is D or Y, X 4 is G or Y, X 5 is D or E, and X 6 is P or S). comprising an amino acid sequence according to a formula selected from the group consisting of: said screening; b. screening a library of second antigen binding domains for binding to a second antigen, said library of second antigen binding domains comprising antigen binding domain members, all of the antigen binding domain members comprising a second antibody heavy chain variable region and a second antibody light chain variable region, at least one second antibody heavy chain variable region having the same sequence as the first antibody heavy chain variable region; c. generating a bispecific antibody comprising the first antigen-binding domain and the second antigen-binding domain; Generation method.

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