Dynamic Human Heavy Chain Antibody Library

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] This disclosure relates to a library containing synthetic polynucleotides encoding antibody heavy chains (e.g., heavy chains of dynamic human antibodies), as well as antibody heavy chains, antibodies, cells, animals, methods, and kits relating thereto. [Background technology]

[0002] Monoclonal antibodies have become extremely useful in a variety of fields, including biological research, medical diagnostics, and pharmaceuticals. Their potential diversity in binding specificity allows for the creation of antibodies with beneficial specificity and capabilities. However, this diversity makes screening a vast number of antibodies to identify one or more with the desired properties a difficult and time-consuming task.

[0003] One method for identifying a target antibody is to screen antibody libraries, such as libraries of cloned B cell sequences, phage display libraries, and yeast display libraries. These libraries allow for screening a large number of antibodies representing numerous unique antibody sequences, enabling the identification of antibodies with desired specific properties, such as binding to a particular target, binding affinity, and selectivity. However, current libraries have certain limitations. Libraries derived from biological sources, such as the human B cell repertoire, are limited to antibody sequences that can be cloned from that source. Synthetic libraries may contain non-natural sequences compared to biologically obtained libraries, but these are also limited by the amount of antibodies that can be synthesized within a given timeframe. Furthermore, very large libraries require more time and a more comprehensive screening approach; otherwise, only a small fraction of the library can actually be screened for the target antibody.

[0004] Therefore, the development of dynamic antibody libraries containing a sufficient set of dynamic units with clearly defined, developable sequence profiles is needed to design and construct potentially highly functionally relevant dynamic antibodies. Such libraries would greatly improve the efficiency of antibody screening, not only in terms of the diversity of antibody-binding sites on antibodies within the library, but also in terms of 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 developability profiles.

[0005] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as is indicated in each individual reference where such reference is specifically and individually referred to. [Overview of the Initiative]

[0006] To satisfy the above and other needs, disclosed herein are heavy chain hypervariable regions (HVRs) and heavy chain variable regions (e.g., V) that enable dynamic human antibodies. HThese are antibody sequences such as (region). These sequences are designed to produce antibodies with highly flexible HVR sequence loops that can bind to targets with high ability and / or recognize multiple useful epitopes and / or cross-react with epitopes shared across different species with low sequence identity (approximately 60% or less). Advantageously, these antibody sequences enable the creation of libraries that contain a large number of useful antibodies in smaller libraries 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 to a wide range of targets, or in some cases, novel antibodies that cross-react with multiple targets of interest. Furthermore, novel concepts and methodologies for designing and constructing dynamic antibody libraries that capture the flexibility of a wide range of three-dimensional structures of antibody binding sites in physically compact libraries using newly identified dynamic units are introduced and implemented herein. Furthermore, results using such antibodies (described below) highlight the possibility of identifying antibodies from these libraries that target three-dimensional structural epitopes and / or evolutionarily conserved sites on a given heterologous antigen with low sequence identity (e.g., less than 60%–70%).

[0007] Accordingly, in one embodiment, 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 the HVR-H1 is given by formula (I): X1TFX2X3YX4IHWV (SEQ ID NO: 198) (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); The amino acid sequence includes an amino acid sequence selected from the group consisting of formula (II):YSIX1SGX2X3WX4WI (SEQ ID NO: 199) (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); and formula (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 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52.

[0008] In another embodiment, provided herein are one or more HVR-H2 amino acid sequences and / or one or more polynucleotides (e.g., synthetic polynucleotides) encoding such amino acid sequences, wherein the HVR-H2 is given by formula (IV):LAX1IX2WX3X4DKX5YSX6SLKSRL (SEQ ID NO: 201) (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); Expression (V): IGX1IX2X3SGSTYYSPSLKSRV (Sequence ID 202) (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y); Expression (VI): IGX1IYX2SGX3TX4YNPSLKSRV (Sequence ID 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); Expression (VII): VSX1ISGX2GX3X4TYYADS VKGRF(Sequence ID 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(Sequence ID 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(Sequence ID The amino acid sequence includes an amino acid sequence that conforms to a formula selected from the group consisting of (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (Sequence ID 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 contains an amino acid sequence that conforms to a formula selected from the group consisting of formulas (IV); (VII); (VIII); (IX); (XI): IGX1IX2X3SGSTYYSPSLKSRV (SEQ ID NO: 208) (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y); (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 (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 includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 53-136 and 159-164.

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

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

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

[0012] In another embodiment, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding antibody heavy chain variable regions 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 formulas (I), (II), and (III). In some embodiments, provided herein are libraries 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 antibody heavy chain variable regions 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 formulas (I), (II), and (III). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of which 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 formulas (I), (II), and (III).

[0013] In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, HVR-H1 includes 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 includes 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 can be combined with any of the prior 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 to 256.

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

[0016] In another embodiment, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding 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 formulas (IV), (V), (VI), (VII), (VII), (VIII), (IX), and (X). In some embodiments, provided herein are libraries comprising polynucleotides (e.g., synthetic polynucleotides), wherein at least one polynucleotide 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 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 formulas (IV), (V), (VI), (VII), (VIII), (IX), and (X). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of which 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 formulas (IV), (V), (VI), (VII), (VIII), (IX), and (X).

[0017] In some embodiments, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding antibody heavy chain variable regions including 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 formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (XIII). In some embodiments, provided herein are libraries 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 antibody heavy chain variable regions including 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 formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (XIII). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of which 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 formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (XIII).

[0018] In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 53-136 and 159-164. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 53-136. In some embodiments, HVR-H2 includes 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 includes 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 includes 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 can be combined with any of the prior 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 to 256.

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

[0021] In another embodiment, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding antibody heavy chain variable regions 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 formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), and (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 HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), and (X). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of which 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 formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), and (X).

[0022] In some embodiments, provided herein are polynucleotides (e.g., synthetic polynucleotides) encoding antibody heavy chain variable regions 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 formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (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 HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (XIII). In some embodiments, provided herein is a library comprising polynucleotides (e.g., synthetic polynucleotides), each of which 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 formulas (I), (II), and (III), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formulas (IV), (VII), (VIII), (IX), (XI), (XII), and (XIII).

[0023] In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, HVR-H1 includes 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 includes 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 includes HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); HVR-H1 and The group is selected from the following: HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XI); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XI); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IX); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XIII).

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

[0026] In some embodiments that can be combined with any of the prior embodiments, the polynucleotides in the library are approximately 6.5*10 4 Less than (for example, approximately 6.5 * 10 4 Less than approximately 5.5*10 4 Less than approximately 2.5*10 4 Less than approximately 1*10 4The polynucleotides in the library (e.g., synthetic polynucleotides) include unique combinations of HVR-H1 and HVR-H2 sequences (less than approximately 6700, less than approximately 6660, less than approximately 5000, less than approximately 2500, less than approximately 1000, less than approximately 690, less than approximately 500, less than approximately 100, less than approximately 50, etc.). In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) include unique combinations of HVR-H1 and HVR-H2 sequences of approximately 62272 or less. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) include unique combinations of HVR-H1 and HVR-H2 sequences of approximately 60928 or less. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) include unique combinations of HVR-H1 and HVR-H2 sequences of approximately 54656 or less. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) include unique combinations of HVR-H1 and HVR-H2 sequences of approximately 6660 or less. In some embodiments, the polynucleotides in the library (e.g., synthetic polynucleotides) contain approximately 690 or fewer unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, at least one of the HVR-H1 and HVR-H2 sequences in the antibody heavy chain variable region takes on multiple conformations when evaluated by structural determination and / or computer modeling.

[0027] In some embodiments that can be combined with any of the prior 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 to 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 a mutation from arginine to lysine 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 polynucleotide in the library encodes a full-length antibody heavy chain. In some embodiments, the library further comprises one or more polynucleotides (e.g., synthetic polynucleotides) encoding antibody light chain variable regions. 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 polynucleotide encoding the antibody light chain variable region comprises at least 1 unique sequence, at least 100 unique sequences, at least 280 unique sequences, at least 10 3 of unique sequences, at least 10 4 of unique sequences, at least 10 5 of unique sequences, at least 10 6A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 It includes a unique sequence. In some embodiments, one or more polynucleotides in the library encoding the antibody light chain variable region encode the full-length antibody light chain.

[0030] In another embodiment, provided herein is a polynucleotide (e.g., synthetic polynucleotide) encoding a plurality of unique antibodies, each antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region of each of the plurality of antibodies comprising the same sequence and encoded by any of the polynucleotides encoding the heavy chain variable region as described above. In some embodiments, provided herein is a library comprising a polynucleotide (e.g., synthetic polynucleotide) encoding a plurality of unique antibodies, each antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region of each of the plurality of antibodies comprising the same sequence and encoded by any of the polynucleotides encoding the heavy chain variable region 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. 257-264, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 265-274. In some embodiments, the light chain variable region of the antibody in the library comprises at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, and at least 10 3 A unique array of at least 10 4 A unique array of at least 10 5 A unique array of at least 10 6 A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 Includes a unique array of the following.

[0032] In another embodiment, provided herein are vectors comprising any of the polynucleotides 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 ten, 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 described above. In some embodiments, at least two of the vectors in the library comprises the polynucleotides described above. In some embodiments, at least 100 of the vectors in the library comprises the polynucleotides described above. In some embodiments, at least 500 of the vectors in the library comprises the polynucleotides described above. In some embodiments, at least 1000 of the vectors in the library comprises the polynucleotides described above. In some embodiments, at least 5000 of the vectors in the library comprises the polynucleotides described above. In some embodiments, at least 6500 of the vectors in the library comprises the polynucleotides described above. In some embodiments, provided herein is a library containing vectors, each of which contains one 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 containing vectors further comprises at least one vector encoding a light chain variable region polypeptide (e.g., at least one, at least two, at least five, at least ten, 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.).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 comprises at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, and at least 10. 3 A unique array of at least 10 4 A unique array of at least 10 5 A unique array of at least 10 6 A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 It codes for a light chain variable region containing a unique sequence.

[0033] In another embodiment, provided herein are cells comprising any of the polynucleotides and / or vectors described above. In some embodiments, provided herein are libraries comprising a population of cells, wherein at least one of the cells in the library (e.g., at least one, at least two, at least five, at least ten, at least 100, at least ten) 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(etc.) comprises any of the polynucleotides and / or vectors described above. In some embodiments, at least two cells in the library contain any of the polynucleotides and / or vectors described above. In some embodiments, at least 100 cells in the library contain any of the polynucleotides and / or vectors described above. In some embodiments, provided herein is a library comprising a population of cells, each of which contains any of the polynucleotides and / or vectors 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 embodiment, provided herein are antibody heavy chain variable regions comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence conforming to a formula selected from the group consisting of formulas (I), (II), and (III). In some embodiments, provided herein are libraries comprising antibody heavy chain variable regions, wherein at least one of the heavy chain variable regions in the library (e.g., at least one, at least two, at least three, at least four, at least five, at least ten, etc.) comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence conforming to a formula selected from the group consisting of formulas (I), (II), and (III). In some embodiments, provided herein is a library comprising antibody heavy chain variable regions, each of which 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 formulas (I), (II), and (III).

[0035] In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, HVR-H1 includes 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 includes 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 can be combined with any of the prior 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 to 256.

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

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

[0040] In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 53-136 and 159-164. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 53-136. In some embodiments, HVR-H2 includes 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 includes 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 includes 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 can be combined with any of the prior 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 to 256.

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

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

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

[0045] In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52. In some embodiments, HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, HVR-H1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136. In some embodiments, HVR-H1 includes 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 includes 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 includes HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); HVR-H1 and The group is selected from the following: HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XI); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XI); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IX); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XIII).

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

[0048] In some embodiments that can be combined with any of the prior 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 to 256.

[0049] In some embodiments, the heavy chain variable region further comprises FW-H1 containing the amino acid sequence of SEQ ID NO: 165. In some embodiments, the heavy chain variable region further comprises FW-H2 containing the amino acid sequence of SEQ ID NO: 166. In some embodiments, the heavy chain variable region further comprises FW-H3 containing the amino acid sequence of SEQ ID NO: 167. In some embodiments, the heavy chain variable region further comprises FW-H4 containing the amino acid sequence of SEQ ID NO: 168. In some embodiments, the heavy chain variable region comprises any combination of at least two (e.g., at least two, at least three, or all four) of FW-H1 containing the amino acid sequence of SEQ ID NO: 165, FW-H2 containing the amino acid sequence of SEQ ID NO: 166, FW-H3 containing the amino acid sequence of SEQ ID NO: 167, and FW-H4 containing the amino acid sequence of SEQ ID NO: 168. In some embodiments, the FW-H3 sequence contains an arginine-to-lysine mutation at R19 of SEQ ID NO: 167. In some embodiments, the heavy chain variable region includes sequences 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 can be combined with any of the prior embodiments, the heavy chain variable region in the library is approximately 6.5*10 4 Less than (for example, approximately 6.5 * 10 4 Less than approximately 5.5*10 4 Less than approximately 2.5*10 4 Less than approximately 1*10 4The library contains unique combinations of HVR-H1 and HVR-H2 sequences (e.g., less than 6700, less than 6660, less than 5000, less than 2500, less than 1000, less than 690, less than 500, less than 100, less than 50, etc.). In some embodiments, the heavy chain variable region in the library contains unique combinations of HVR-H1 and HVR-H2 sequences of about 62272 or less. In some embodiments, the heavy chain variable region in the library contains unique combinations of HVR-H1 and HVR-H2 sequences of about 60928 or less. In some embodiments, the heavy chain variable region in the library contains unique combinations of HVR-H1 and HVR-H2 sequences of about 54656 or less. In some embodiments, the heavy chain variable region in the library contains unique combinations of HVR-H1 and HVR-H2 sequences of about 6660 or less. In some embodiments, the heavy chain variable region in the library contains approximately 690 or fewer unique combinations of HVR-H1 and HVR-H2 sequences. In some embodiments, at least one of the HVR-H1 and HVR-H2 sequences in the antibody heavy chain variable region takes on multiple three-dimensional structures when evaluated by structural determination and / or computer modeling.

[0051] In another embodiment, 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 described herein. In some embodiments, provided herein is a library comprising antibody heavy chain variable regions and antibody light chain variable regions, wherein at least one of the antibody heavy chain variable regions in the library (e.g., at least one, at least two, at least five, at least ten, at least 100, etc.) is any of the heavy chain variable regions described herein. In some embodiments, provided herein is a library 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 described herein. 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 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 region in the library contains at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, and at least 10 3 A unique array of at least 10 4 A unique array of at least 10 5 A unique array of at least 10 6 A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 Includes a unique array of the following.

[0052] In another embodiment, provided herein is an antigen-binding domain comprising an antibody heavy chain variable region, wherein the antigen-binding domain comprises any of the antibody heavy chain variable regions described herein. In some embodiments, provided herein is a library comprising antigen-binding domains comprising antibody heavy chain variable regions, wherein at least one of the antigen-binding domains in the library (e.g., at least one, at least two, at least five, at least ten, at least 100, etc.) comprises any of the heavy chain variable regions described herein. In some embodiments, provided herein is a library comprising antigen-binding domains comprising antibody heavy chain variable regions, wherein each of the antigen-binding domains in the library comprises any of the heavy chain variable regions described herein. In some embodiments, the antigen-binding domain further comprises an antibody light chain variable region comprising 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 antigen-binding domains in the library, including the light chain variable region, consist of at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, and at least 10 3 A unique array of at least 10 4 A unique array of at least 10 5 A unique array of at least 10 6 A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 It includes a light chain variable region containing a unique sequence.

[0053] In another embodiment, provided herein is an antibody comprising an antibody heavy chain variable region, the antibody comprising any of the antibody heavy chain variable regions described herein. In some embodiments, provided herein is a library comprising antibodies, the at least one of the antibodies in the library (e.g., at least one, at least two, at least five, at least ten, at least 100, etc.) comprising any of the antibody heavy chain variable regions described herein. In some embodiments, provided herein is a library comprising antibodies, each of the antibodies in the library comprising any of the heavy chain variable regions described herein. In some embodiments, the antibody further comprises an antibody light chain variable region comprising 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, antibodies containing light chain variable regions in a library include at least one unique sequence, at least 100 unique sequences, at least 280 unique sequences, and at least 10 3 A unique array of at least 10 4 A unique array of at least 10 5 A unique array of at least 10 6 A unique array of at least 10 7 A unique array of at least 10 8 A unique array of, or at least about 10 9 It includes a light chain variable region containing a unique sequence.

[0054] In some embodiments that can be combined with any of the prior embodiments, the antibody is approximately 6.5*10 4 It contains a unique combination of HVR-H1 and HVR-H2 sequences less than 5.5*10. In some embodiments that can be combined with any of the prior embodiments, the antibody is approximately 5.5*10 4The antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than 100. In some embodiments, the antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than or equal to about 62272. In some embodiments, the antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than or equal to about 60928. In some embodiments, the antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than or equal to about 54656. In some embodiments, the antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than or equal to about 6660. In some embodiments, the antibody contains a unique combination of HVR-H1 and HVR-H2 sequences less than or equal to about 690. In some embodiments, at least one of the HVR-H1 and HVR-H2 sequences in the antibody heavy chain variable region takes on multiple three-dimensional forms when evaluated by structural determination and / or computer modeling.

[0055] In some embodiments that can be combined with any of the prior embodiments, the antibody is approximately 10 -7 ~about 10 -11 The antibody binds to at least one target at 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 embodiment, provided herein are polypeptides (e.g., scaffold polypeptides) containing one or more HVRs of the Disclosure (e.g., one or more, two or more, three or more, four or more, five or more, etc.). In some embodiments, provided herein are libraries containing polypeptides, where at least one of the polypeptides in the library (e.g., at least one, at least two, at least five, at least ten, 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.) contains one or more HVRs of the Disclosure. In some embodiments, provided herein are libraries containing polypeptides, where each of the polypeptides in the library contains one or more HVRs of the Disclosure. In some embodiments, the polypeptide comprises HVR-H1 comprising an amino acid sequence selected from any HVR-H1 sequence as described herein (e.g., formulas (I), (II), and (III); and HVR-H1 according to a formula selected from the group consisting of SEQ ID NOs: 1-52 and 137-158). In some embodiments, the polypeptide comprises HVR-H2 comprising an amino acid sequence selected from any HVR-H2 sequence as described herein (e.g., formulas (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), and (XIII); and HVR-H2 according to a formula selected from the group consisting of SEQ ID NOs: 53-136 and 159-164). In some embodiments, the polypeptide comprises 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 having an amino acid sequence selected from any HVR-L1 sequence described herein (e.g., SEQ ID NOs. 257-264).In some embodiments, the polypeptide comprises an HVR-L3 having an amino acid sequence selected from any HVR-L3 sequence described herein (e.g., SEQ ID NOs. 265-274). In some embodiments, the polypeptide comprises two or more (e.g., two or more, three or more, four or more, or all five) of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and / or HVR-L3 sequences described herein. In some embodiments, provided herein are a polynucleotide encoding any of the above polypeptides and a library comprising the polynucleotide.

[0057] In another embodiment, provided herein is a phage having at least one polypeptide on its surface, wherein the at least one polypeptide comprises one of the antibody heavy chain variable regions described herein. In some embodiments, the at least one polypeptide is one of the antigen-binding domains described herein. In some embodiments, provided herein is a library of phages, wherein at least one of the phages in the library (e.g., at least one, at least two, at least five, at least ten, 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.) has at least one polypeptide on its surface comprising one of the antibody heavy chain variable regions described herein. In some embodiments, at least one phage in the library has at least one polypeptide on its surface comprising one of the antigen-binding domains described herein. In some embodiments, provided herein is a library comprising phages, each of which comprises at least one polypeptide having on its surface one of the antibody heavy chain variable regions described herein. In some embodiments, the at least one polypeptide is one of the antigen-binding domains described herein.

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

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

[0060] In another embodiment, provided herein is a method for screening target-binding polypeptides, comprising incubating one of the libraries containing polypeptides described herein (e.g., a library of antigen-binding domains, a library of antibodies, a library of phages, etc.) with a target, and selecting one or more polypeptides from the target-binding library.

[0061] In another embodiment, provided herein is a method for preparing an antibody library, comprising the steps of (a) selecting one, two, or three heavy chain HVRs having multiple three-dimensional structures, and (b) assembling polynucleotide sequences to produce 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 one of the heavy chain variable region sequences described herein. In some embodiments, each of the multiple antibody heavy chain variable region sequences is one of the heavy chain variable region sequences described herein.

[0062] In another embodiment, provided herein are methods for preparing polypeptides (e.g., heavy chain variable regions, antibody heavy chains, antibodies, scaffold polypeptides, etc.), comprising culturing cells containing any of the above-described polynucleotides, polynucleotide libraries, vectors, and / or vector libraries to produce polypeptides. In some embodiments, the polypeptides are recovered from the cultured cells and further purified.

[0063] In another embodiment, provided herein is a method for producing a bispecific antibody containing two antibody heavy chain variable regions and two identical light chain variable regions, comprising: (a) screening for a first antigen-binding domain that binds to a first antigen, wherein the first antigen-binding domain comprises a first antibody heavy chain variable region and a first antibody light chain variable region, 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, wherein the second antigen-binding domain comprises a 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) producing a bispecific antibody containing the first antigen-binding domain and the second antigen-binding domain.

[0064] In another embodiment, 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 the same sequence as the first heavy chain variable region sequence, wherein each of the first and second heavy chain variable regions comprises one 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, the second light chain comprising a second light chain variable region, and both the first and second light chains each comprising kappa C L Domain (for example, Hitokappa C) L (including domain). 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, the second light chain comprising a second light chain variable region, and both the first and second light chains each comprising lambda C L Domain (for example, human-lambda C L(including domains). 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 kappa C L Domain (for example, Hitokappa C) L The second light chain includes the domain, and the second light chain variable region and lambda C L Domain (for example, human-lambda C L (including domains). 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 lambda C L Domain (for example, human-lambda C L The domain includes the second light chain, and the second light chain variable region and kappa C L Domain (for example, Hitokappa C) L Includes domains.

[0065] In another embodiment, provided herein are kits comprising any of the polynucleotides, polynucleotide libraries, vectors, and / or vector libraries (or any cells or populations 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, 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 embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure are further described below. [Brief explanation of the drawing]

[0067] [Figure 1]Figure 1A shows the amino acid entropy plot of the VH domain for each residue number. Entropy was calculated using the VH structures of 113 human antibodies. Figure 1B shows the definition of the 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] This shows the affinity measurements of fabs that were confirmed to bind to antigens TAGT-1 to TAGT-12. [Figure 2B] The melting temperature (Tm) measurements of fabs confirmed to bind to antigens TAGT-1 to TAGT-12 are shown. [Modes for carrying out the invention]

[0068] This 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 include HVR sequences designed to generate highly flexible loops for obtaining more effective substrate binding and / or specificity to multiple substrates of interest. These HVR sequences enable 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 skilled in the art and refer to conventional methodologies, such as 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 (FMAusubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor 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 It is commonly used, employing widely adopted methodologies such as those described in *Press, 1999*; *The Antibodies* (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995); and *Cancer: Principles and Practice of Oncology* (VT DeVita et al., eds., JBLippincott Company, 1993).

[0070] II. Definition Before detailing this disclosure, please understand that this disclosure is not limited to any particular composition or biological system, and is naturally subject to change. Furthermore, please understand that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0071] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly indicated by the context. Thus, for example, a reference to "a molecule" may optionally include a combination of two or more such molecules.

[0072] As used herein, the term “approximately” refers to the normal margin of error for each value, as readily understood by those skilled in the art. References to values ​​or parameters “approximately” herein include (and are described) embodiments relating to the value or parameter itself.

[0073] It should be understood that the aspects and embodiments of the Disclosure described herein include "including," "consisting of," and "essentially derived from" the 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 scFv) insofar as they exhibit the desired biological activity.

[0075] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). H and V L When these two antibodies pair together, a single antigen-binding site is formed. For the structures and properties of different antibody classes, see, for example, 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] The light chain (L) derived from any vertebrate species can be assigned to one of two distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of its 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 chain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain denoted as alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"), respectively. The γ and α classes are further classified 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 various immunoglobulin classes are known, for example, Abbas et al., Cellular and Molecular Immunology, 4 th An overview is provided in the ed. (WBSaunders Co., 2000).

[0077] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is called "V H It is sometimes called "V". The variable domain of the light chain is "V L These domains are sometimes referred to as "[domain name]". These domains are generally the most variable part of the antibody and contain the antigen-binding site.

[0078] The terms "Kabat variable domain residue numbering" or "Kabat amino acid position numbering," and their variations, refer to the numbering system used by Kabat et al. (cited above) to organize the heavy chain or light chain variable domains of antibodies. When using this numbering system, the actual linear amino acid sequence may contain fewer amino acids depending on the shortening of the FR or HVR of the variable domain, or additional amino acids depending on insertions into the FR or HVR. For example, a heavy chain variable domain may contain one amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and multiple inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues for a given antibody can be determined by alignment of the antibody sequence with a homologous region of a "standard" Kabat numbering sequence.

[0079] The Kabat numbering system is generally used when referring to residues in the variable domain (generally light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al. (above)). The "EU index according to Kabat" refers to the residue numbering of human IgG1 EU antibodies.

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

[0081] The term “full-length antibody” (the terms “intact” antibody 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” antibody heavy chain 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. Specifically, whole antibodies include those having a heavy chain and a light chain containing an Fc region. The constant domain may be the constant domain of the natural sequence (e.g., the constant domain of the human natural sequence) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0082] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population. That is, the individual antibodies in that population are identical except for any spontaneous mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to polyclonal antibody preparations, which typically contain various antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with this disclosure can be prepared by various techniques, including, for example, hybridoma methods (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, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (e.g., see U.S. Patent Nos. 4,816, 567), and phage display methods (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al. 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.See Methods 284(1-2):119-132(2004), and techniques for producing human antibodies or human-like antibodies in animals possessing a human immunoglobulin locus or part or all of a human immunoglobulin gene encoding a human immunoglobulin sequence (e.g., WO1998 / 24893; WO1996 / 34096; WO1996 / 33735; WO1991 / 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 No. 5,545,807; No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425 and No. 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 (See Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0083] As used herein, “hypervariable regions (HVRs)” refer to regions of an antibody domain whose sequence is hypervariable and / or forms structurally defined loops. Generally, antibodies contain six HVRs, three located in VH(H1, H2, H3) and three in VL(L1, L2, L3). See, for example, 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 consists of three HVR and four framework (FW) regions arranged from the amino terminus to the carboxyl 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 HVR in the exemplary antibody heavy chain variable domain shown in Figure 1B (as used herein) 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, “library” refers to a collection of two or more entities that share a common class. For example, a library containing polynucleotides may refer to a collection of two or more polynucleotides. The term “library” is used herein in its broadest sense and specifically includes sublibraries, which may or may not be combined.

[0085] As used herein, "unique" refers to a member of a set that is distinct from the other members of that set. For example, a unique antibody derived from a library encoding multiple polynucleotides that encode antibodies may be an antibody that has a specific sequence not shared by other antibodies encoded by that library. In practice, it should be understood that a "unique" member physically realized in a library may exist in more than one copy. For example, a library may contain multiple "unique" antibodies, and one or more of these "unique" antibody molecules may arise 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 may refer to the variety of antibodies containing unique sequences present in the library.

[0087] The terms “polypeptide,” “protein,” and “peptide” are used herein without distinction and may refer to polymers of two or more amino acids.

[0088] As used herein without distinction, “polynucleotide” or “nucleic acid” refers to polymers of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reactions. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure may be conjugated before or after the assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may include post-synthesis modifications, such as conjugations to labels. Other modification types include, for example, "caps," substitution of one or more native nucleotides by analogs, internucleotide modifications, such as modifications by uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and modifications by charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties, such as modifications containing proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications by intercalators (e.g., acridine, psoralens, etc.), modifications containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, modifications by modification bonds (e.g., α-anomeric nucleic acids, etc.), and the unmodified form of polynucleotides. Furthermore, any of the hydroxyl groups normally present in the sugar may be replaced by, for example, a phosphonic acid group or a phosphate group, protected by a standard protecting group, activated to generate further binding to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' OH groups may be phosphorylated or substituted with amines or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may also include analogous forms of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro- or 2'-azid-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 methylriboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, a substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl, or an araldyl. Not all links within the polynucleotide are to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0089] Cells (e.g., cells or populations of cells containing synthetic polynucleotides or libraries of synthetic polynucleotides) include individual cells or cell cultures that can be recipients of or are recipients of a vector(s) for incorporating polynucleotide inserts. Host cells include offspring of a single host cell, which may not necessarily be completely identical to the original parent cell (morphologically or in terms of genomic DNA complementation) due to spontaneous, accidental, or intentional mutations. Host cells include cells transfected in vivo with polynucleotides(s) (e.g., synthetic polynucleotides encoding the antibody heavy chain variable regions of this disclosure).

[0090] "Non-human animals" refers to any animal not classified as human, such as livestock, farm or zoo animals, sports animals, pet animals (dogs, horses, cats, cattle, etc.), and animals used for research. Research animals may include, 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 macaques, crab-eating macaques, chimpanzees, etc.). In a preferred embodiment, the animal is an antibody-producing animal.

[0091] III. Antibody Libraries and Library Preparation Certain aspects of this disclosure relate to polynucleotides, for example, antibody heavy chain variable regions (V H ) or light chain variable region (V L The present disclosure relates to a library of polynucleotides encoding HVR-H1, HVR-H2, and HVR-H3. The library may contain one or more polynucleotides encoding heavy chain variable regions, the HVR-H1 and HVR-H2 being any of the HVR-H1 and / or HVR-H2 described herein.

[0092] In some embodiments, the library of this disclosure has fewer unique heavy chain HVR sequences and / or unique V sequences than a typical antibody library. H The libraries contain sequences. Advantageously, such libraries can provide sufficient diversity for identifying antibodies that bind to one or more of a large number of antigens of interest, and the smaller library size also enables more efficient screening. In some embodiments, the libraries of the Disclosure contain or consist of polynucleotides containing about 10,000, about 9,000, about 8,000, or about 7,000 unique combinations of HVR-H1 and HVR-H2 sequences. In certain embodiments, the libraries of the Disclosure contain or consist of polynucleotides containing about 6,600 or fewer unique combinations of HVR-H1 and HVR-H2 sequences.

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

[0094] In some embodiments, one or more polynucleotides encode an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is (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)YSIX1SGX The amino acid sequence contains a formula selected from the group consisting of 2X3WX4WI (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) (Sequence ID 199); and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200). In some embodiments, one or more polynucleotides encode an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 is (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) (Sequence ID 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence ID 202); (Formula VI)IGX 1IYX2SGX3TX4YNPSLKSRV(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)(Sequence ID 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)(Sequence ID 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)(Sequence ID 205);The amino acid sequence contains an amino acid sequence that conforms to a formula selected from the group consisting of (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) (Sequence ID 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) (Sequence ID 207). In some embodiments, HVR-H2 contains an amino acid sequence that conforms to a formula selected from the group consisting of (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210). In some embodiments, one or more polynucleotides encode an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is (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) (Sequence ID 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) (Sequence ID 199);and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200) contain an amino acid sequence that conforms to a formula selected from the group, and HVR-H2 contains an amino acid sequence that conforms to a formula selected from the group, and (Formula IV)LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, and X3 is A, D, S, or Y (Formula V) IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, R, S, or Y) (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) (Formula VIII) IGX1INPNX2GX3TX4YAQ KFQGRV (wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N) (Sequence ID 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) (Sequence ID 206);The amino acid sequence includes an amino acid sequence that conforms to a formula selected from the group consisting of (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) (Sequence ID 207). In some embodiments, one or more polynucleotides encode an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is derived from (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) (Sequence ID 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) (Sequence ID 199); and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200) HVR-H2 contains an amino acid sequence that follows a formula selected from the group consisting of (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210). In some embodiments, one or more polynucleotides from the library are present in a vector (e.g., an expression vector or a display vector).

[0095] In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, 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, and a small number of polynucleotides. 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 include HVR-H1, HVR-H2, and HVR-H3. The antibody heavy chain variable region is coded, and the HVR-H1 is (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) (Sequence ID 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) (Sequence ID 199);and an amino acid sequence according to a formula selected from the group consisting of (Formula III) FSLSTX1GVX2VX3WI (where X1 is G or S, X2 is A or G, 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, 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, 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, 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, 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, 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, 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, X6 is P or S) (SEQ ID NO: 207); HVR-H2 comprising an amino acid sequence according to a formula selected from the group consisting of; and / or about 6.5*10 of the polynucleotides; 4 less than (e.g., about 6.5*10 4 less than, about 5.5*10 4 less than, about 2.5*10 4Less than approximately 1*10 4Numbers less than, approximately less than 6700, approximately less than 6660, approximately less than 5000, approximately less than 2500, approximately less than 1000, approximately less than 690, approximately less than 500, approximately less than 100, approximately less than 50, etc., 62272 or less, 60928 or less, 54656 or less, or 6660 or less encode the antibody heavy chain variable region including HVR-H1, HVR-H2, and HVR-H3, and the HVR-H1 is (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) (sequence) The amino acid sequence includes a formula selected from the group consisting of (Formula 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) (Sequence ID 199); and (Formula III) FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200), and (Formula IV) LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X (Formula V) IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, R, S, or Y, X2 is H or Y, and X3 is P or T) (Sequence 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) (Sequence No. 202); (Formula VI) IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, R, S, or Y, X2 is H or Y, and X3 (wherein X1 is N or S, and X4 is N or Y) (Sequence code 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) (Sequence code 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) (Sequence code 205);HVR-H2 comprising an amino acid sequence conforming to a formula selected from the group consisting of (Formula IX)IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N) (SEQ ID NO: 206); and (Formula X)VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S) (SEQ ID NO: 207). In some embodiments, one or more polynucleotides of the library are in a vector (e.g., an expression vector or a display vector).

[0096] In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, 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, and a small number of polynucleotides. 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 include HVR-H1, HVR-H2, and HVR-H3. The antibody heavy chain variable region is coded, and the HVR-H1 is (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) (Sequence ID 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) (Sequence ID 199);and an amino acid sequence according to a formula selected from the group consisting of (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 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, about 5.5*10 4 less than, about 2.5*10 4 less than, about 1*10 4Numbers less than, approximately less than 6700, approximately less than 6660, approximately less than 5000, approximately less than 2500, approximately less than 1000, approximately less than 690, approximately less than 500, approximately less than 100, approximately less than 50, etc., 62272 or less, 60928 or less, 54656 or less, or 6660 or less encode an antibody heavy chain variable region containing HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is (Formula I)X1TFX2X3YX4IHWV (wherein X1 is F or Y) (wherein X1 is S or T, X2 is H or Y, X3 is D, G, N, or S, and X4 is A, G, or W) (Sequence 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) (Sequence No. 199); and (Formula III) FSLSTX1GVX2VX3WI (wherein X1 is G or S, X The amino acid sequence includes a formula selected from the group consisting of (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (Formula XI)IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S) HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S)

[0097] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198), and (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, and X5 is R, S, (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence 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) (Sequence 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) (Sequence No. 203); (Formula VII)VSX1ISGX2GX3X4TYY ADSVKGRF (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) (Sequence ID 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) (Sequence ID 205); (Formula IX)IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W The antibody heavy chain variable region comprises HVR-H2 having an amino acid sequence that follows a formula selected from the group consisting of (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) (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) (Formula X)

[0098] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198), and (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3T The antibody heavy chain variable region comprises HVR-H2 containing an amino acid sequence that conforms to a formula selected from the group consisting of X4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (Sequence ID 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (Sequence ID 210).

[0099] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 19), and (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, and X5 is R, S, (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence 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) (Sequence 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) (Sequence No. 203); (Formula VII)VSX1ISGX2GX3X4TYY ADSVKGRF (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) (Sequence ID 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) (Sequence ID 205); (Formula IX)IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W The antibody heavy chain variable region comprises HVR-H2 having an amino acid sequence that follows a formula selected from the group consisting of (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) (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) (Formula X)

[0100] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199), and (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3 The antibody heavy chain variable region comprises HVR-H2 containing an amino acid sequence that follows a formula selected from the group consisting of TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210).

[0101] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200), and (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) (Sequence ID 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence ID 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) (Sequence ID 203); (Formula VII)VSX1ISGX2GX3X4TYYADSVKGR F (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) (Sequence ID 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) (Sequence ID 205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W, and X2 The antibody heavy chain variable region comprises HVR-H2 having an amino acid sequence that follows a formula selected from the group consisting of (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) (Formula X)

[0102] In some embodiments, the polynucleotides in the library include HVR-H1 containing an amino acid sequence according to formula FSLTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200), and (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XI)IGX1IYX2SGX3TX4YNPSL The antibody heavy chain variable region comprises HVR-H2 containing an amino acid sequence that follows a formula selected from the group consisting of KSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210).

[0103] 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 to 52 and 137 to 158.

[0104] In some embodiments, the polynucleotide library encodes an antibody 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.

[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 formulas (I), (II), and (III), or HVR-H2 comprises an amino acid sequence selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), and (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 to 52, or 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, 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, wherein 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 this disclosure. In some embodiments, the heavy chain variable region includes HVR-H1, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2, which includes 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, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2, which includes 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, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H3, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, the heavy chain variable region includes HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H1, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158; HVR-H2, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164; and HVR-H3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.In some embodiments, the heavy chain variable region includes HVR-H1, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52; HVR-H2, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136; and HVR-H3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.

[0108] In a particular embodiment, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); HVR-H The group is selected from the following: HVR-H2 containing the amino acid sequence of formula (1) and formula (VI); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XI); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XI); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IX); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XIII).

[0109] In a particular embodiment, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of SEQ ID NO: 157 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; HVR-H1 includes the amino acid sequence of SEQ ID NO: 1 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 122; HVR-H1 includes the amino acid sequence of SEQ ID NO: 138 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; and HVR-H1 includes the amino acid sequence of SEQ ID NO: 154 and SEQ ID NO: 63 HVR-H2 containing amino acid sequences; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 161; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 145 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 128; HVR-H1 containing the amino acid sequence of SEQ ID NO: 22 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 61; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 63 HVR-H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 153 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 155 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 67; HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 100; HVR-H1 containing the amino acid sequence of SEQ ID NO: 51 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 123 VR-H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 139 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; HVR-H1 containing the amino acid sequence of SEQ ID NO: 8 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 126; HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 129; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 124; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 130;The group is selected from the following: HVR-H1 containing the amino acid sequence of SEQ ID NO: 150 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 132; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 12 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 82; HVR-H1 containing the amino acid sequence of SEQ ID NO: 149 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 117; and HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 include the amino acid sequence of SEQ ID NO: 26 and HVR-H2 including the amino acid sequence of SEQ ID NO: 53; HVR-H1 including the amino acid sequence of SEQ ID NO: 151 and HVR-H2 including the amino acid sequence of SEQ ID NO: 53; HVR-H1 including the amino acid sequence of SEQ ID NO: 34 and HVR-H2 including the amino acid sequence of SEQ ID NO: 63; HVR-H1 including the amino acid sequence of SEQ ID NO: 50 and HVR-H2 including the amino acid sequence of SEQ ID NO: 162; HVR-H1 including the amino acid sequence of SEQ ID NO: 158 and HVR-H2 including the amino acid sequence of SEQ ID NO: 104; HVR-H1 including the amino acid sequence of SEQ ID NO: 5 and HVR-H2 including the amino acid sequence of SEQ ID NO: 121; and HVR-H1 including the amino acid sequence of SEQ ID NO: 6 and SEQ ID NO: 116 HVR-H2 containing amino acid sequences; HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 121; HVR-H1 containing the amino acid sequence of SEQ ID NO: 17 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 101; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 114; HVR-H1 containing the amino acid sequence of SEQ ID NO: 29 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; HVR-H1 containing the amino acid sequence of SEQ ID NO: 152 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 89;HVR-H1 containing the amino acid sequence of SEQ ID NO: 157 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 94; HVR-H1 containing the amino acid sequence of SEQ ID NO: 48 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 58; HVR-H1 containing the amino acid sequence of SEQ ID NO: 50 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 89; HVR-H1 containing the amino acid sequence of SEQ ID NO: 50 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 163; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 160; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 87; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 92; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 93; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 97; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 103; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 164; HVR-H1 containing the amino acid sequence of SEQ ID NO: 137 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 54; HVR-H1 containing the amino acid sequence of SEQ ID NO: 3 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 127; HVR-H1 containing the amino acid sequence of SEQ ID NO: 4 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 85; HVR-H1 containing the amino acid sequence of SEQ ID NO: 4 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; Sequence number HVR-H1 containing the amino acid sequence of sequence number 139 and HVR-H2 containing the amino acid sequence of sequence number 109; HVR-H1 containing the amino acid sequence of sequence number 139 and HVR-H2 containing the amino acid sequence of sequence number 121; HVR-H1 containing the amino acid sequence of sequence number 8 and HVR-H2 containing the amino acid sequence of sequence number 120; HVR-H1 containing the amino acid sequence of sequence number 140 and HVR-H2 containing the amino acid sequence of sequence number 131; HVR-H1 containing the amino acid sequence of sequence number 141 and HVR-H2 containing the amino acid sequence of sequence number 116;The following are selected from the group consisting of HVR-H1 containing the amino acid sequence of SEQ ID NO: 142 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 159; HVR-H1 containing the amino acid sequence of SEQ ID NO: 143 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 116; HVR-H1 containing the amino acid sequence of SEQ ID NO: 144 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 121; HVR-H1 containing the amino acid sequence of SEQ ID NO: 146 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; HVR-H1 containing the amino acid sequence of SEQ ID NO: 147 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 133; HVR-H1 containing the amino acid sequence of SEQ ID NO: 148 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; and HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing 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 to 256. In some embodiments, the heavy chain variable region includes 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 with 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 includes variable region heavy chain framework sequences arranged in close proximity between HVRs, as follows: (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 as follows: FW-H1 is EVQLVESGGGLVQPGGSLRLSCAASG (Sequence ID 165), FW-H2 is RQAPGKGLEW (Sequence ID 166), FW-H3 is TISRDNSKNTLYLQLNSLRAEDTAVYYC (Sequence ID 167), FW-H4 is WGQGTLVTVSS (Sequence ID 168).

[0112] In some embodiments, the heavy chain variable region includes an alternative FW-H3 sequence having an arginine-to-lysine mutation at R19 in SEQ ID NO: 167. In some embodiments, one, two, three, or four of the framework sequences are FW-H1 in SEQ ID NO: 165, FW-H2 in SEQ ID NO: 166, SEQ ID NO: 167 or FW-H3 having an arginine-to-lysine mutation at R19, and FW-H4 in 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., HVR-L1, HVR-L2, and HVR-L3). In some embodiments, the antibody light chain variable region includes HVR-L1, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 257-264. In some embodiments, the antibody light chain variable region includes HVR-L3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 265-274. In some embodiments, the antibody light chain variable region includes HVR-L1, which contains 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, and 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 It contains multiple polynucleotides that encode unique sequences of the antibody light chain variable region. In some embodiments, the library contains at least 10 3 It contains multiple polynucleotides that encode unique sequences of the antibody light chain variable region. In some embodiments, the library contains at least 10 5 It contains multiple polynucleotides that encode unique sequences of the antibody light chain variable region. In some embodiments, the library contains at least 10 9 The library contains multiple polynucleotides encoding unique sequences of the antibody light chain variable region. In other embodiments, the library contains a single polynucleotide encoding the antibody light chain variable region. In some embodiments, the library contains 1 to about 10 3It contains multiple polynucleotides encoding a unique sequence of the antibody light chain variable region. In some embodiments, the antibody light chain variable region is any of the antibody light chain variable regions found in the patent applications filed under agent reference numbers 69540-3000100, 69540-2000140, and / or 69540-2000100 (their disclosures are incorporated herein by reference in their entirety). In some embodiments, the antibody light chain variable region includes any of the HVR-L1, HVR-L2, and / or HVR-L3 sequences found in the patent applications filed under agent reference numbers 69540-3000100, 69540-2000140, and / or 69540-2000100 (their disclosures are incorporated herein by reference in their entirety).

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

[0115] In some embodiments, the library contains multiple polynucleotides encoding multiple unique antibodies. In some embodiments, each antibody includes a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region of each of the multiple antibodies includes the same sequence and comprises HVR-H1, HVR-H2, and HVR-H3. In some embodiments, at least one or at least two of HVR-H1 and HVR-H2 are the HVR-H1 sequences of the Disclosure (e.g., X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198); YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, X 4 is A, D, G, N, S, or T) (Sequence ID 199); and FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200); and Sequence IDs 1-52 and 137-158), and the HVR-H2 sequences of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, and X3 is A, D (wherein X1 is A, D, E, S, or Y, X2 is S, or Y, and X6 is P or T) (Sequence ID 201); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S, or Y, and X3 is H, or Y) (Sequence ID 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 (wherein X1 is Y) (Sequence code 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) (Sequence code 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) (Sequence code 205);IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N) (Sequence ID 206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S) (Sequence ID 207); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 The amino acid sequences include those selected from (wherein X1 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210); and SEQ ID NOs: 53-136 and 159-164). The heavy chain HVR sequences described herein may be included in any combination in the library of this disclosure, which also includes polynucleotides encoding one or more light chain variable regions.

[0116] In some embodiments, the library of the Disclosure comprises one or more vectors encoding one or more polynucleotides of the Disclosure (e.g., synthetic polynucleotides).

[0117] Further provided herein are methods for preparing a library, for example, by preparing and assembling polynucleotide sequences (e.g., synthetic polynucleotides) of the library of this disclosure. Further provided herein are methods for preparing a library, for example, by selecting one, two, or three heavy chain HVRs (e.g., one or two heavy chain HVRs of this disclosure) having sequences having multiple three-dimensional structures, and assembling the polynucleotide sequences to produce 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 HVR-H1 and / or HVR-H2 are HVR-H1 sequences of the present disclosure (e.g., X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198); YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199); and FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200); (and sequence numbers 1-52 and 137-158), and the HVR-H2 sequences of the present disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T) (sequence number 201); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (sequence number 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) (sequence number 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) (Sequence ID 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 (Sequence code 205);IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N) (Sequence code 206);VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (wherein X1 is K or R, X2 is A or T, and X3 is D The amino acid sequences include those selected from (wherein X1 is Y, X4 is G or Y, X5 is D or E, X6 is P or S) (Sequence ID 207); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, X3 is H or Y) (Sequence ID 208); IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, X4 is N or Y) (Sequence ID 209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is P or S) (Sequence ID 210); and Sequence IDs 53-136 and 159-164). ;

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

[0119] A set of polynucleotides encoding antibody light chain and / or heavy chain variable regions can be cloned into any vector suitable for the expression of part or all of the light chain sequence or heavy chain sequence. In some embodiments, the polynucleotides cloned into the vector can be fused to all or part of a viral coat protein (i.e., to produce a fusion protein), enabling the production of part or all of the light chain sequence or heavy chain sequence presented on the surface of a particle or cell. Several types of vectors are available, such as phagemide vectors, and can be used in the implementation of this disclosure. Phagemide vectors generally contain a variety of components, including a promoter, signal sequence, phenotypic selection gene, origin of replication site, and other necessary components known to those skilled in the art. In some embodiments, a set of polynucleotides encoding antibody light chain 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. An exemplary vector is described in U.S. Patent Application Publication US20160145604. In some embodiments, the vector is a display vector comprising, from 5' to 3', a polynucleotide encoding an amino acid sequence to be presented on a surface (e.g., the surface of a phage, bacterium, yeast, or mammalian cell), a restriction site, a second polynucleotide encoding a surface peptide having the ability to be presented on the surface, and a second restriction site. In some embodiments, the second polynucleotide encodes a phage coat protein, yeast outer wall protein, bacterial outer membrane protein, cell surface tether domain, or adapter, or a truncated or derivative thereof. In a particular embodiment, 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 for shuttle display across these. In some embodiments, the amino acid sequence and surface peptide, when expressed, are presented on the surface as a fusion protein.In some embodiments, the vector further includes a fusion tag on the 5' side of the first restriction site or on the 3' side of the second restriction site.

[0120] Certain aspects of this disclosure relate to a population of cells containing the vector(s) described herein. The light and / or heavy chains of antibodies encoded by polynucleotides generated by the techniques described herein or any other preferred techniques can be expressed and screened to identify antibodies having a desired structure and / or activity. Antibody expression can be carried out, for example, using cell-free extracts (e.g., ribosome display), phage display, prokaryotic cells (e.g., bacterial display), or eukaryotic cells (e.g., yeast display). In some embodiments, the cells are bacterial cells, yeast cells, or mammalian cells. Methods for transfecting bacterial cells, yeast cells, or mammalian cells are known in the art and are described in the references cited herein. The expression of polypeptides (e.g., antibody chains) in these cell types (e.g., expression from the libraries of this disclosure) and the screening of the antibodies of interest are described below in detail.

[0121] Alternatively, polynucleotides can be expressed in E. coli expression systems such as those described by Pluckthun and Skerra (Meth. Enzymol., 1989, 178:476; Biotechnology, 1991, 9:273). Mutant proteins can be expressed by secretion in culture medium and / or bacterial cytoplasm, as described by Better and Horwitz, Meth. Enzymol., 1989, 178:476. In some embodiments, V H and V LThe single domain encoding the signal is conjugated to the 3' end of a sequence encoding a signal sequence, such as ompA, phoA, or pelB signal sequences, respectively (Lei et al., J. Bacteriol., 1987, 169:4379). The fusion of these genes is assembled into a dicistrone construct, which allows these fusions to be expressed from a single vector, secreted into the periplasm of E. coli, where they refold and are recovered in an active form (Skerra et al., Biotechnology, 1991, 9:273). For example, an antibody heavy chain gene can be expressed together with an antibody light chain gene to produce an antibody or antibody fragment.

[0122] In other embodiments, the antibody sequence is expressed on the membrane surface of a prokaryote, such as E. coli, using secretory signaling and lipid-forming moieties, as described, for example, 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, Jeong et al., PNAS, 2007, 104:8247, or by other anchoring methods, as described, for example, Mazor et al., Nature Biotechnology, 2007, 25:563.

[0124] Mammalian cells, such as myeloma cells (e.g., NS / O cells), hybridoma cells, Chinese hamster ovary (CHO) cells, and human embryonic kidney (HEK) cells, as well as other higher eukaryotic cells, can also be used for the expression of the antibodies of this disclosure. Typically, antibodies expressed in mammalian cells are designed to be secreted into the culture medium or expressed on the surface of the cells. The antibody or antibody fragment may be expressed, for example, as an intact antibody molecule or as individual V H and V LIt can be generated as a fragment, a Fab fragment, a single domain, or as a single strand (scFv).

[0125] In other embodiments, antibodies can be selected using mammalian cell display (Ho et al., PNAS, 2006, 103:9637). In some embodiments illustrated above and below, antibodies can be selected, for example, by fusion to all or part of a virus-coding protein (i.e., generation of a fusion protein) using phage display, after generating part or all of a light chain sequence or heavy chain sequence presented on the surface of a particle or cell.

[0126] Certain aspects of this disclosure relate to non-human animals comprising the polynucleotide library of this disclosure. For example, the non-human animals of this disclosure may have their genomes modified to include polynucleotides encoding the heavy chain variable regions of this disclosure. In non-limiting examples, transgenic mice are created that include heavy chain immunoglobulin loci modified to express one or more of the heavy chain variable regions of this disclosure. In some embodiments, the transgenic animals (e.g., mice) express antibodies or heavy chains encoded by polynucleotides. Techniques for modifying one or more immunoglobulin loci in non-human animals are known in the art (e.g., methods used to create Xenomouse®).

[0127] Screening of antibodies derived from the libraries of this disclosure can be carried out by any suitable means known in the art. For example, binding activity can be evaluated by standard immunoassays and / or affinity chromatography. Screening of antibodies of this disclosure for catalytic function (e.g., proteolytic function) can be achieved using standard assays, such as hemoglobin plaque assays. Determination of antibody binding affinity to a target can be assayed in vitro using various known techniques, such as BIACORE® instruments that measure the binding rate of antibodies to a given target or antigen based on surface plasmon resonance, or Bio-Layer Interferometry (BLI) using the ForteBio Octet® RED96 platform (Pall Life Sciences), as illustrated below. In vivo assays can be performed using one of a number of animal models and then, if necessary, tested in humans. Cell-based biological assays are also contemplated. Antibodies or antigen-binding fragments can be further selected for functional activity, such as antagonist activity or agonist activity. Exemplary screening methods are described herein. For example, in some embodiments, the binding affinity between a fab fragment(s) and one or more targets(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 is tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in host cells 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 kinetic buffer.

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

[0129] IV. Antibodies and Antibody Production Provided herein are antibodies defined and selected from the libraries described herein. Certain aspects of this disclosure relate to the HVR, variable regions containing the HVR, and / or polynucleotides encoding them of the light or heavy chain of an antibody. In some embodiments, the HVR 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 (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) (Sequence ID 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) (Sequence ID 199); and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200). In some embodiments, the heavy chain variable region includes HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H2 is (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) (Sequence ID 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence ID 202); (Formula VI)IGX1IYX2SGX3TX 4YNPSLKSRV(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)(Sequence ID 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)(Sequence ID 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)(Sequence ID 205);The amino acid sequence contains an amino acid sequence that conforms to a formula selected from the group consisting of (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) (Sequence ID 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) (Sequence ID 207). In some embodiments, HVR-H2 contains an amino acid sequence that conforms to a formula selected from the group consisting of (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XII)IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210). In some embodiments, the heavy chain variable region includes HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is (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) (Sequence ID 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) (Sequence ID 199);and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200) comprising an amino acid sequence that conforms to a formula selected from the group, and (Formula IV)LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, and X4 (wherein X1 is D or G, X5 is R, S, or Y, and X6 is P or T) (Sequence 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) (Sequence 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) (Sequence ID 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) (Sequence ID 204); (Formula VIII) IGX1INPNX2GX3TX4YAQKF QGRV (wherein X1 is I, R, or W, X2 is F or R, X3 is D, G, or S, and X4 is K or N) (Sequence ID 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) (Sequence ID 206);HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of (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) (Sequence ID 207). In some embodiments, the heavy chain variable region includes HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is a formula selected from the group consisting of (Formula I)X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (Sequence ID 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) (Sequence ID 199); and (Formula III)FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 200). HVR-H2 containing an amino acid sequence that conforms to a formula selected from the group consisting of (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 208); (Formula XII)IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210).

[0131] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198), and (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, and X5 is R, S, or Y) (wherein X6 is P or T) (Sequence code 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence code 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) (Sequence code 203); (Formula VII)VSX1ISGX2GX3X4T YYADSVKGRF (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) (Sequence ID 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) (Sequence ID 205); (Formula IX)IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of (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) (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) (Formula X)

[0132] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198), and (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)IGX1IYX2SG HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of X3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (Sequence ID 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (Sequence ID 210).

[0133] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199) and (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, and X5 is R, S, or Y) (wherein X6 is P or T) (Sequence code 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence code 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) (Sequence code 203); (Formula VII)VSX1ISGX2GX3X4 TYYADSVKGRF (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) (Sequence ID 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) (Sequence ID 205); (Formula IX)IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of (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) (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) (Formula X)

[0134] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199), and (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)IGX1IYX2S HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of GX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (Sequence ID 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (Sequence ID 210).

[0135] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200) and (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 (Sequence number 201); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence number 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) (Sequence number 203); (Formula VII)VSX1ISGX2GX3X4TYYADSVK GRF (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) (Sequence ID 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) (Sequence ID 205); (Formula IX) IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, and HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of (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) (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) (Formula X)

[0136] In some embodiments, the heavy chain variable region comprises HVR-H1 containing an amino acid sequence according to formula FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200), and (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)IGX1IYX2SGX3TX4YN HVR-H2 comprising an amino acid sequence that conforms to a formula selected from the group consisting of PSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, X3 is N or S, and X4 is N or Y) (Sequence ID 209); and (Formula XII)VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (Sequence ID 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 contain the amino acid sequences listed in Table 1 below. Table 1: Heavy chain HVR sequence TIFF0007856719000001.tif250170TIFF0007856719000002.tif251170TIFF0007856719 000003.tif251170TIFF0007856719000004.tif251170TIFF0007856719000005.tif86170

[0138] In some embodiments, the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein 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 to 256.

[0139] In some embodiments, provided herein are antibody heavy chains having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and / or HVR-H2 are any of the HVR-H1 and / or HVR-H2 described herein. In some embodiments, HVR-H1 comprises an amino acid sequence selected from any HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198); YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199); and FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200); as well as SEQ ID NOs: 1-52 and 137-158). In some embodiments, HVR-H2 is any HVR-H2 of the Disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T) (Sequence ID 201); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence ID 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) (Sequence ID 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) (Sequence ID 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) (Sequence ID 205);IGX1IX2PSX3GX4TX5YAQKFQGRV (wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N) (Sequence ID 206); VGRIX1SKX2X3GX4TTX5YAAX6VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, X5 is D or E, and X6 is P or S) (Sequence ID 207); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 The amino acid sequence includes an amino acid sequence selected from (wherein X1 is S or Y, and X3 is H or Y) (Sequence ID 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) (Sequence ID 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) (Sequence ID 210); and Sequence IDs 53-136 and 159-164.

[0140] In some embodiments, provided herein are antibody heavy chains having heavy chain variable regions 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 are antibody heavy chains 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 are antibody heavy chains 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 formulas (I), (II), and (III), or HVR-H2 comprises an amino acid sequence selected from the group consisting of formulas (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), and (XIII). In some embodiments, provided herein are antibody heavy chains 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 to 52, or 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, 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 are antibody heavy chains having a heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 comprises an amino acid sequence selected from SEQ ID NOs. 1-52 and 137-158, and HVR-H2 comprises an amino acid sequence selected from SEQ ID NOs. 53-136 and 159-164. In some embodiments, HVR-H1 comprises an amino acid sequence selected from SEQ ID NOs. 1-52, and HVR-H2 comprises an amino acid sequence selected from SEQ ID NOs. 53-136.

[0144] In a particular embodiment, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); HVR-H The group is selected from the following: HVR-H2 containing the amino acid sequence of formula (1) and formula (VI); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII).In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XI); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XI); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (XI). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IV); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (IX); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (X); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (X). In some embodiments, HVR-H1 and HVR-H2 are selected from the group consisting of HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (XIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (XIII); and HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the 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 this disclosure. In some embodiments, the heavy chain variable region includes HVR-H1, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H2, which includes 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, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52, and HVR-H2, which includes 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, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and HVR-H3, which includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256. In some embodiments, the heavy chain variable region includes HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136, and HVR-H3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256. In some embodiments, the heavy chain variable region includes HVR-H1, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158; HVR-H2, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-136 and 159-164; and HVR-H3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 223-256.In some embodiments, the heavy chain variable region includes HVR-H1, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52; HVR-H2, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136; and HVR-H3, which contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 223 to 256.

[0146] In a particular embodiment, the heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of SEQ ID NO: 157 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; HVR-H1 includes the amino acid sequence of SEQ ID NO: 1 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 122; HVR-H1 includes the amino acid sequence of SEQ ID NO: 138 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; and HVR-H1 includes the amino acid sequence of SEQ ID NO: 154 and SEQ ID NO: 63 HVR-H2 containing amino acid sequences; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 161; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 145 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 128; HVR-H1 containing the amino acid sequence of SEQ ID NO: 22 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 61; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 63 HVR-H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 153 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 155 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 67; HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 100; HVR-H1 containing the amino acid sequence of SEQ ID NO: 51 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 123 VR-H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 139 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; HVR-H1 containing the amino acid sequence of SEQ ID NO: 8 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 126; HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 129; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 124; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 130;The group is selected from the following: HVR-H1 containing the amino acid sequence of SEQ ID NO: 150 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 132; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 12 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 82; HVR-H1 containing the amino acid sequence of SEQ ID NO: 149 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 117; and HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 134. In some embodiments, HVR-H1 and HVR-H2 include the amino acid sequence of SEQ ID NO: 26 and HVR-H2 including the amino acid sequence of SEQ ID NO: 53; HVR-H1 including the amino acid sequence of SEQ ID NO: 151 and HVR-H2 including the amino acid sequence of SEQ ID NO: 53; HVR-H1 including the amino acid sequence of SEQ ID NO: 34 and HVR-H2 including the amino acid sequence of SEQ ID NO: 63; HVR-H1 including the amino acid sequence of SEQ ID NO: 50 and HVR-H2 including the amino acid sequence of SEQ ID NO: 162; HVR-H1 including the amino acid sequence of SEQ ID NO: 158 and HVR-H2 including the amino acid sequence of SEQ ID NO: 104; HVR-H1 including the amino acid sequence of SEQ ID NO: 5 and HVR-H2 including the amino acid sequence of SEQ ID NO: 121; and HVR-H1 including the amino acid sequence of SEQ ID NO: 6 and SEQ ID NO: 116 HVR-H2 containing amino acid sequences; HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 121; HVR-H1 containing the amino acid sequence of SEQ ID NO: 17 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 101; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 114; HVR-H1 containing the amino acid sequence of SEQ ID NO: 29 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; HVR-H1 containing the amino acid sequence of SEQ ID NO: 152 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 89;HVR-H1 containing the amino acid sequence of SEQ ID NO: 157 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 94; HVR-H1 containing the amino acid sequence of SEQ ID NO: 48 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 58; HVR-H1 containing the amino acid sequence of SEQ ID NO: 50 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 89; HVR-H1 containing the amino acid sequence of SEQ ID NO: 50 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 163; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 160; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 87; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 92; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 93; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 97; HVR-H1 containing the amino acid sequence of sequence number 158 and HVR-H2 containing the amino acid sequence of sequence number 103; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 164; HVR-H1 containing the amino acid sequence of SEQ ID NO: 137 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 54; HVR-H1 containing the amino acid sequence of SEQ ID NO: 3 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 127; HVR-H1 containing the amino acid sequence of SEQ ID NO: 4 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 85; HVR-H1 containing the amino acid sequence of SEQ ID NO: 4 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; Sequence number HVR-H1 containing the amino acid sequence of sequence number 139 and HVR-H2 containing the amino acid sequence of sequence number 109; HVR-H1 containing the amino acid sequence of sequence number 139 and HVR-H2 containing the amino acid sequence of sequence number 121; HVR-H1 containing the amino acid sequence of sequence number 8 and HVR-H2 containing the amino acid sequence of sequence number 120; HVR-H1 containing the amino acid sequence of sequence number 140 and HVR-H2 containing the amino acid sequence of sequence number 131; HVR-H1 containing the amino acid sequence of sequence number 141 and HVR-H2 containing the amino acid sequence of sequence number 116;The following are selected from the group consisting of HVR-H1 containing the amino acid sequence of SEQ ID NO: 142 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 159; HVR-H1 containing the amino acid sequence of SEQ ID NO: 143 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 116; HVR-H1 containing the amino acid sequence of SEQ ID NO: 144 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 121; HVR-H1 containing the amino acid sequence of SEQ ID NO: 146 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; HVR-H1 containing the amino acid sequence of SEQ ID NO: 147 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 133; HVR-H1 containing the amino acid sequence of SEQ ID NO: 148 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; and HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing 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 to 256. In some embodiments, the heavy chain variable region includes a sequence selected from sequence numbers 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 with a sequence selected from sequence numbers 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 includes variable region heavy chain framework sequences arranged in close proximity between HVRs, as follows: (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 as follows: FW-H1 is EVQLVESGGGLVQPGGSLRLSCAASG (Sequence ID 165), FW-H2 is RQAPGKGLEW (Sequence ID 166), FW-H3 is TISRDNSKNTLYLQLNSLRAEDTAVYYC (Sequence ID 167), FW-H4 is WGQGTLVTVSS (Sequence ID 168).

[0149] In some embodiments, the heavy chain variable region includes an alternative FW-H3 sequence having an arginine-to-lysine mutation at R19 in SEQ ID NO: 167. In some embodiments, one, two, three, or four of the framework sequences are FW-H1 in SEQ ID NO: 165, FW-H2 in SEQ ID NO: 166, SEQ ID NO: 167 or FW-H3 having an arginine-to-lysine mutation at R19, and FW-H4 in SEQ ID NO: 168.

[0150] In some embodiments, further provided herein are antibodies comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region of the Disclosure, and the light chain comprises any light chain variable region known in the Art (including, for example, 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 to 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 to 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 to 264, and HVR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 265 to 274. In some embodiments, the antibody light chain includes any of the antibody light chain variable regions found in the patent applications filed under agent reference numbers 69540-3000100, 69540-2000140, and / or 69540-2000100 (their disclosures being incorporated herein by reference in their entirety). In some embodiments, the antibody light chain includes a light chain variable region containing any of the HVR-L1, HVR-L2, and / or HVR-L3 sequences found in the patent applications filed under agent reference numbers 69540-3000100, 69540-2000140, and / or 69540-2000100 (their disclosures being incorporated herein by reference in their entirety).

[0151] IgG-derived scaffolds such as Fab and single-stranded Fv (scFv), as well as stabilized Fv or scFv, are designed and prepared to possess the ability to specifically recognize and firmly bind to antigens. Alternative protein scaffolds, or non-IgG-like scaffolds, are being explored for similar applications. Several protein families with non-Ig structures, such as protein A, fibronectin, ankyrin repeats, adnectin, afibody, anticarin, DARPin, engineered Kunitz inhibitors or lipocalin, and cyclic and polycyclic peptides, can be given novel 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 novel alternative binding reagents are collectively referred to as engineered protein scaffolds, demonstrating the fact that robust native protein structures are used to modify existing binding sites for a given target or to give novel binding sites using the dynamic binding motifs or units presented herein. These protein scaffolds offer practical advantages over antibodies or their recombinant fragments, often including improved stability in microbial expression systems and higher production yields. Since these novel binding proteins are obtained through biomolecular engineering processes to achieve dense target binding activity, they can also be subjected to further selection schemes focusing on other desired properties (e.g., solubility, thermal stability, protease resistance, etc.). As a result, engineered protein scaffolds are attractive for numerous applications in biotechnology and biomedical research, particularly for multispecific binding motifs. Efforts to generate such alternative binding proteins with beneficial properties are directed toward therapeutic uses, as well as clinical applications, with a particular emphasis on the structure and function of biomolecules.

[0152] In some embodiments, further provided herein are one or more polypeptides comprising one or more HVRs described herein (e.g., IgG-derived scaffold polypeptides (Fab, single-stranded Fv, and stabilized Fv, etc.) or non-IgG-derived scaffold polypeptides (protein A, fibronectin, ankyrin repeat, adonectin, afibody, anticarin, DARPin, engineered Kunitz inhibitors or lipocalin, cyclic and polycyclic peptides, etc.)). In some embodiments, the polypeptide comprises an HVR-H1 comprising an amino acid sequence selected from any HVR-H1 sequence of the present disclosure (e.g., X1TFX2X3YX4IHWV (wherein X1 is F or Y, X2 is S or T, X3 is D, G, N, or S, and X4 is A, G, or W) (SEQ ID NO: 198); YSIX1SGX2X3WX4WI (wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T) (SEQ ID NO: 199); and FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (SEQ ID NO: 200); as well as SEQ ID NOs: 1-52 and 137-158). In some embodiments, the polypeptide is any HVR-H2 of the Disclosure (e.g., LAX1IX2WX3X4DKX5YSX6SLKSRL (wherein X1 is L or R, X2 is D or Y, X3 is A, D, S, or Y, X4 is D or G, X5 is R, S, or Y, and X6 is P or T) (SEQ ID NO: 201); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (SEQ ID NO: 202); IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, R, S, or Y, X2 is H or Y, X3 is N or S, and X4 is N or Y) (SEQ ID NO: 203);VSX1ISGX2GX3X4TYYADSVKGRF (wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T) (Sequence ID 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) (Sequence code 205);IGX1IX2PSX3GX4TX5YAQKFQGRV(wherein X1 is I, R, or W, X2 is S or Y, X3 is G or S, X4 is D, G, or S, and X5 is K or N)(Sequence code 206);VGRIX1SKX2X3GX4TTX5YAAX6VKGRF(wherein X1 is K or R, X2 is A or T, and X3 is D or Y) Yes, where X4 is G or Y, X5 is D or E, and X6 is P or S) (Sequence ID 207); IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (Sequence ID 208); IGX1IYX2SGX3TX4YNPSLKSRV (wherein X1 is D, E, or S, X2 is H or Y, and X3 is N The polypeptide comprises HVR-H2 comprising an amino acid sequence selected from (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: 209); and VGRIX1SKX2X3GX4TTEYAAX5VKGRF (wherein X1 is K or R, X2 is A or T, X3 is D or Y, X4 is G or Y, and X5 is P or S) (SEQ ID NO: 210); and SEQ ID NOs. 53-136 and 159-164). In some embodiments, the polypeptide comprises 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 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 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, the polypeptide comprises two or more (e.g., two or more, three or more, four or more, or all five) of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and / or HVR-L3 sequences described herein. In some embodiments, the polypeptide comprises two of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, and / or HVR-L3 sequences described herein, where these two are 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, which 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-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, which 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] In some embodiments, further provided herein are antibody fragments or scFv comprising the light chain variable region and heavy chain variable region of the present disclosure.

[0155] In some embodiments, the antibodies or antibody fragments of the Disclosure bind 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, the antibodies or antibody fragments of the Disclosure bind to about 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 It binds to at least one target or at least two targets with an equilibrium dissociation constant (Kd) of M or less. In some embodiments, the antibody or antibody fragment of this disclosure is about 10 -7 ~about 10 -11 It binds to at least one target or at least two targets at the equilibrium dissociation constant (Kd) of M. Exemplary assays for determining binding affinity are described and illustrated below (see, for example, the ForteBio assay in Example 4 below).

[0156] In some embodiments, the antibodies or antibody fragments of the Disclosure have a melting temperature (Tm) of at least 60°C. For example, in some embodiments, the antibodies or antibody fragments of the Disclosure have 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 antibodies or antibody fragments of the Disclosure have 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 illustrated below (see, for example, the DSF assay in Example 4 below).

[0157] The antibodies of this disclosure may be produced, for example, using the recombinant methods and compositions described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids encoding any of the antibodies described herein are provided. Such nucleic acids are the V of the antibody. L Amino acid sequence including and / or V H It may encode an amino acid sequence containing (e.g., the light and / or heavy chain of an antibody). In some embodiments, one or more vectors containing such nucleic acids (e.g., expression vectors) are provided herein. In some embodiments, host cells containing such nucleic acids are provided. In one such embodiment, the host cell contains (1) the V of the antibody L Amino acid sequence containing and antibody V H (2) A vector containing nucleic acids encoding an amino acid sequence including (2) an antibody L A first vector and antibody containing nucleic acids encoding an amino acid sequence including HThe present invention comprises a second vector containing a nucleic acid encoding an amino acid sequence including (e.g., transformed with the vector). In some embodiments, the host cell is a eukaryote, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In some embodiments, a method for producing an antibody is provided, comprising culturing a host cell containing the nucleic acid encoding the antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell medium).

[0158] To recombinantly produce the antibodies of this disclosure, for example, the nucleic acid encoding the antibody described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of the antibody).

[0159] Suitable host cells for cloning or expressing antibody coding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254 (description of antibody fragment expression in E. coli)). After expression, antibodies can be isolated from bacterial cell paste in the soluble fraction and further purified.

[0160] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody coding vectors. These include fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of antibodies with a partially or completely human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0161] Host cells suitable 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. Along with insect cells, numerous baculovirus strains have been identified that can be used, in particular, for transfection of Spodoptera frugiperda cells.

[0162] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0163] Vertebrate cells can 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: monkey kidney CV1 cell line (COS-7) transformed by SV40; human embryonic kidney cell line (293 cells or 293 cells as described, e.g., Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); e.g., Mather et al., Annals These include TRI cells; MRC5 cells; and FS4 cells, as described in NYAcad.Sci.383:44-68 (1982). 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, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKCLo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

[0164] Bispecific antibodies with identical / common / single heavy chains Further provided herein are bispecific antibodies having identical heavy chain variable regions (for example, 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 (for example, Hitokappa C) L The second light chain includes the domain, and the second light chain is lambda C L Domain (for example, human-lambda C L (Domain) includes. Kappa C L Domain and Lambda C L Methods for producing and / or purifying bispecific antibodies containing domains are known in the art (see, for example, Fischer et al. (2015), Nat.Commun. 6:6113; US20140179547). For example, 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 kappa C L a) a first light chain containing a domain, and c) a second light chain variable region and lambda C L A second light chain containing a domain (e.g., kappa C) L The constant region of the second light chain, which includes the domain, is lambda C L A bispecific antibody containing (with the domain replaced by) and can be constructed and expressed (e.g., cloned into one or more expression vectors and expressed in one or more suitable host cells). The bispecific IgG (e.g., both kappa and lambda C) constructed and obtained in this way can be constructed and expressed (e.g., both kappa and lambda C). L Bispecific IgG (containing the 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. This removes free light chains and other impurities. Next, KappaC is purified using KappaSelect affinity resin. L Captures IgG containing the domain and lambda C L Single-specific IgG containing only the domain and light chain is removed by column flow-through. Finally, pure bispecific kappa-lambda bodies are recovered using LambdaFabSelect affinity resin, and kappa C that does not bind to the resin is removed. LIsolate from monospecific IgG having light chains containing only the domain. Alternatively, purify bispecific common heavy chain IgG (e.g., IgG as described above) with protein A and isolate each light chain C based on one or more differences in the biophysical properties of the different light chains (e.g., different molecular weight, different isoelectric point (pI), etc.). L They can be separated 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, wherein the bispecific antibody comprises a first binding domain that binds to a first target or antigen and includes 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 includes a second antibody light chain variable region and a second antibody heavy chain variable region, wherein the second antibody heavy chain variable region has the same sequence as 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 part of a first antibody heavy chain that includes 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 the second antibody heavy chain including the second heavy chain variable region and the second heavy chain constant region (e.g., including CH1, hinge, CH2, and CH3). In some embodiments, the first antibody light chain variable region is a portion of the first antibody light chain including the first light chain variable region and the first light chain constant region. In some embodiments, the second antibody light chain variable region is a portion of the second antibody light chain including the second light chain variable region and the second light chain constant region. In some embodiments, the first and second antibody heavy chains have the same sequence as the heavy chains of this disclosure.

[0166] Further provided herein are methods for producing a bispecific antibody having identical heavy chain variable regions of the present disclosure (for example, having two light chain variable regions having 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 includes 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 includes a second antibody light chain variable region and a second heavy chain variable region of the present disclosure, wherein the second antibody heavy chain variable region has the same sequence as the first antibody heavy chain variable region sequence; and (c) producing a bispecific antibody that includes a light chain variable region containing the amino acid sequence of the first antibody light chain variable region, a light chain variable region containing the amino acid sequence of the second antibody light chain variable region, a heavy chain variable region containing the amino acid sequence of the first antibody heavy chain variable region sequence, and a heavy chain variable region containing 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 derived from the library of this disclosure.

[0167] In some embodiments, the bispecific antibodies described herein may have further specificity. For example, one of the antigen-binding sites or target-binding sites of the bispecific antibody may specifically bind to two or more targets.

[0168] Methods for producing / creating 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)). Since the heavy and light chains of immunoglobulins are in random combinations, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography steps, but this is quite cumbersome and results in low product yields. Similar methods are disclosed in WO93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0169] V. Kit In another embodiment, provided herein is a kit comprising a library of polynucleotides of the present disclosure. In some embodiments, the kit further includes a package insert, which includes instructions for expressing, modifying, screening, or otherwise using the library, for example, to identify an antibody HVR or variable region of interest. In some embodiments, the kit further includes one or more buffers for storing, transporting, transfecting, or otherwise using, for example, one or more polynucleotides (e.g., synthetic polynucleotides). In some embodiments, the kit further includes one or more containers for storing one or more polynucleotides. In some embodiments, the kit further includes one or more vectors for transfecting, for example, one or more polynucleotides into host cells. [Examples]

[0170] This disclosure will be better understood by referring to the following examples. However, the examples should not be construed as limiting the scope of this disclosure. The examples and embodiments described herein are for illustrative purposes only, and various variations or modifications taking them into account should be understood to those skilled in the art to be conscientious of and included within the spirit and scope of this application and the appended claims.

[0171] Example 1: Identification of the minimum set of dynamic motifs in the hypervariable region To understand the variability of antibody variable domains at the structural level, we developed an algorithm to map the geometric alignment of antibody variable domains and, based on that geometric alignment, calculate structural and sequence entropy. This approach combines the classical theory of antibody diversity, determined by the well-established V(D)J gene rearrangement process, with dynamic unit-based structural diversity (template-oriented stereochemistry selection by Linus Pauling; see, for example, 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), enabling sampling of a nearly infinite epitope space through the selection and fitting of antibody binding sites. As an example, we used this algorithm to analyze the structural and sequence variability of 113 human antibody variable heavy chain domains in high-resolution crystal structures. The entropy was calculated and plotted for all positions in the variable heavy chain domain (Figure 1A; structural entropy is shown as a thick line, and sequence entropy as a dotted line). Using the results obtained from the calculation of structural and sequence entropy based on geometric alignment, hypervariable (HVR) regions were identified, and key positions on these variable regions were identified. For comparison, the HVR (defined by the method described above) and CDR (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. While variability was generally low when assessed by structural alignment, numerous sites / regions exhibited dramatic structural changes, suggesting that these variable regions may play a crucial role in antibody function. Furthermore, some of these hypervariable regions possessed multiple three-dimensional structures, demonstrating high flexibility. Identifying highly variable residue regions provided a more comprehensive overview of the conservation and variability of antibody variable domains, which can be utilized in new antibody design. This identification of dynamic motifs made it possible to cover a wide range of structural diversity with a small number of amino acid sequences. A remarkable advantage of this approach to antibody design was the ability to cover a wide range of antibody structural diversity by utilizing a more limited number of dynamic motifs in the variable regions, providing these antibodies with broad flexibility that can enable binding to multiple target antigens. Therefore, the dynamic heavy chain library was constructed using a single human germline sequence or a human germline-derived sequence for the invariant residues, while the hypervariable regions HVR_H1 and HVR_H2 utilized a limited number of dynamic motifs (10 6 , 10 10 By using (or compared to) the wide range of structural variability identified in these two regions, we were able to grasp the extent of structural variability.

[0173] Example 2: Construction of a common heavy chain library Construction of a heavy chain library To begin construction of the heavy chain library, three groups of degenerate oligonucleotides were designed for the variable region HVR-H1 based on the formulas shown in Table 2, yielding 112 unique HVR-H1 sequences. For the variable region HVR-H2, seven groups of degenerate oligonucleotides were designed based on the formulas shown in Table 2, yielding 565 unique HVR-H2 sequences. The synthesized degenerate oligonucleotides were converted to double-stranded DNA using the following protocol: 0.75 μL of 0.2 μM template oligonucleotides were mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mixture, 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 minutes, then subjected to 14 cycles (15 seconds at 96°C, 15 seconds at 60°C, and 6 seconds at 72°C), followed by extension at 72°C for 3 minutes. 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] Double-stranded VH_vr1 and VH_vr2 were joined via duplicate sequences at their 5' or 3' ends. The protocol used was as follows: 20 ng of VH_vr1 and 20 ng of VH_vr2 templates were mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mixture, 1 μL of 100 μM F_1999 primer, 1 μL of 100 μM R_2003 primer, 0.5 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and water (up to 50 μL). The mixture was preheated at 96°C for 5 minutes, followed by 14 cycles (15 seconds at 96°C, 15 seconds at 60°C, and 10 seconds at 72°C), and then extended at 72°C for 3 minutes. Next, these PCR fragments were purified by gel electrophoresis (GENEray Gel Extraction kit), digested with BspEI and BstBI (Thermo Scientific), and then cloned into filter vector FTV014 digested with the same two enzymes. Using this ligation mixture, DH10B cells were transformed by electroporation, and more than 10 times the calculated diversity of colonies were recovered for plasmid preparation. The purified plasmids constituted library VH-vr12. Table 2: Formulas for the HVR-H1 and HVR-H2 design variant arrays TIFF0007856719000006.tif244170TIFF0007856719000007.tif33170

[0175] The following protocol will 10 5Hundreds of degenerate oligonucleotides encoding VH_vr3 with sequence diversity close to that of VH_vr3 were designed, synthesized, and converted to double-stranded DNA. 0.75 μL of 0.2 μM template oligonucleotides were mixed with 10 μL of 5x PrimeSTAR buffer, 4 μL of dNTP mixture, 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 minutes, followed by 14 cycles (15 seconds at 96°C, 15 seconds at 60°C, and 6 seconds at 72°C), and then extended at 72°C for 3 minutes. 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 subsequently cloned into filter vector FTV012 digested with the same two restriction enzymes. Using this ligation mixture, DH10B cells were transformed by electroporation, and a number of colonies exceeding 10 times the calculated diversity was collected 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), 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 product (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 (maximum 88.5 μL), heated at 95°C for 3 minutes, and annealed for 65 cycles (30 seconds each) with the temperature decreasing by 1°C each 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 product was first digested with NotI to purify the DNA fragment (QIAquick® PCR Purification Kit), and then further digested with XhoI. The digested product was then ligated with T4 DNA ligase (Thermo Scientific). After purification by ethanol precipitation, this ligated product was used to transform DH10B cells by electroporation. The purified plasmids constituted the library VH-vr123. These constructs each shared the same framework region, namely 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] Plasmid mixtures of the two heavy chain libraries described above were digested with PvuI and Acc65I and ligated to the phagemid vector Fad40, which had also been digested with the same two restriction enzymes. Using this ligation mixture, DH10B cells were transformed, and the resulting libraries were purified, quantified, and stored for the assembly of a complete phagemid library.

[0178] Building a VL Library To begin constructing 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 using 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, 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 minutes, then subjected to 14 cycles (15 seconds at 96°C, 15 seconds at 60°C, and 6 seconds at 72°C), followed by extension at 72°C for 3 minutes. VL_vr1 was amplified using the primer pair F_2898(TACTTATGTAGGCGATCGGGTCACCATCACCTGC)(SEQ ID NO: 217) and R_2898(CGGAGCTTTTCCTGGTTTCTGTTGATAC)(SEQ ID NO: 218), while VL_vr2 was amplified using the primer pair F_2013(GAAACCAGGAAAAGCTCCGAAG)(SEQ ID NO: 219) and R_2013(CGTCCCGGAACCGGATCCAGAGAAGCGAG)(SEQ ID NO: 220).

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

[0180] The following protocol was used to design, synthesize, and convert to double-stranded DNA 22 groups of degenerate oligos encoding VL_vr3. 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, followed by 14 cycles (15 seconds at 96°C, 15 seconds at 60°C, and 6 seconds at 72°C), and then extended at 72°C for 3 minutes. Next, the double-stranded DNA encoding VL_vr3 was purified by gel electrophoresis (GENEray Gel Extraction kit), digested with PstI and Acc65I (Thermo Scientific), and then cloned into filter vector FTV013 digested with the same two restriction enzymes. Using this ligation mixture, DH10B cells were transformed by electroporation, and a number of colonies exceeding 10 times the calculated diversity was recovered for plasmid preparation. The purified plasmid constituted the VL-vr3 library.

[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). This fragment was then cloned into the VL-vr12 library plasmid mixture digested with the same two restriction enzymes. Using this ligation product, DH10B cells were transformed by electroporation, and a number of colonies exceeding 10 times the calculated diversity was collected for plasmid preparation. The purified plasmid constituted the library VL-vr123. The vr123 insert of the library plasmid VL-vr123 was then transferred to the phagemide vector Fad40 using the restriction enzymes PvuI and Acc65I. The size of the library containing Fad40-vr123 was 4*10 7 It reached its goal.

[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 library plasmid and the Fad40-vr123 library plasmid were digested with BspEI and SpeI (Thermo Scientific). The DNA fragment encoding the heavy chain from the VH-vr123 library was cloned into a vector backbone derived from the Fad40-vr123 library. Ligation products were desalted before rolling circle amplification (RCA) (QIAquick® PCR Purification Kit (QIAGEN)). 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 (maximum 88.5 μL), and annealed by heating at 95°C for 3 minutes, followed by 65 cycles of 1°C each (30 seconds 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 product was first digested with NotI to purify the DNA fragments (QIAquick® PCR Purification Kit), and then further digested with Acc65I. The digested product was then ligated with T4 DNA ligase (Thermo Scientific). After purification by ethanol precipitation, ER2738 cells were transformed with the ligation product by electroporation. A total of 1.4*10 cells were obtained from the plate. 10 Colonies were collected (2xYT, 1% glucose, 100 μg / mL ampicillin) and a DPL6 library was prepared.

[0183] Example 3: Screening of a common heavy chain library for isolation of the target antibody Preparation of dynamic library phagemid particles To prepare dynamic library phagemide particles for antigen panning, 5.0 liters of ER2738 cells (described in Example 2 above) containing the dynamic library were mixed with a starting OD of 0.1. 600 The cells were then seeded in a medium containing 2xYT, 2% glucose, 100 μg / mL ampicillin, and 12.5 μg / mL tetracycline. The culture medium was shaken at 250 rpm to an OD of 0.6-0.8. 600 The cells were grown at 37°C until the desired concentration was reached. Then, at a multiple infection degree (MOI) of 10, the cells were infected with M13KO7 helper phage for 30 minutes 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 collected by centrifugation at 10,000 rpm for 15 minutes and filtered through a 0.45 μm low-binding membrane filter (Corning). Phagemide particles were then precipitated from the filtered supernatant using PEG / NaCl and resuspended in PBS. Additional rounds of precipitation with PEG / NaCl and subsequent resuspending in PBS were performed. The phage concentration was adjusted to OD. 268 Measurement (OD 268 One unit is approximately 1 * 10 13 The concentration was determined (assumed to be phage particles / mL) and confirmed by plaque assay. Library phagemide particles were stored in 20% glycerol at -80°C.

[0184] Phage Library Panning Antigen proteins at concentrations of 1–30 μg / ml were coated onto Maxisorp strips (Thermo Scientific, catalog number 446469) overnight at 4°C. Multiple antigen wells were prepared for each library. The coated wells were first blocked with 5% milk-containing PBS at room temperature for 1–2 hours and then washed with PBS. Subsequently, 1,100 μL / well of phagemid particle solution (typically 1–5*10 in 2% milk-containing PBS) was added. 12The phages were added to four parallel wells and incubated for 1-2 hours. The wells were then washed several times with PBS, increasing the concentration of Tween 20 (from 0.1% to 0.3%), and finally washed with PBS alone. The bound phagemide particles were eluted from the wells with 100 μL of 0.2 M glycine-HCl at room temperature for 10 minutes. The eluted phages were immediately neutralized with 18 μL of 1 M Tris-HCl (pH 9.1).

[0185] Alternatively, phagemid library panning was performed using Dynabeads (M280, streptavidin, Invitrogen, catalog number 60210) from KingFisher (Thermo Scientific) according to the manufacturer's instructions. 300 μL of Dynabeads were washed with PBS and incubated with biotinylated anti-human Fc for 20 minutes at room temperature. The beads were then blocked in PBS with 5% BSA for 1 hour at room temperature. Fc fusion antigens (70-100 pmol) were captured by incubation at room temperature for 1 hour. The beads were then washed once with PBS and 1 mL of phage library solution (typically 5*10 in 5% BSA-PBS) was added. 12 ~1*10 13 The beads were incubated with phage particles for 1-2 hours. The beads were then washed several times with PBS / Tween (0.1%-0.3%) and PBS, and the bound phages were eluted from the beads with 100 μL of 0.2 M glycine-HCl at room temperature for 10 minutes. The eluted phages 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 for each antigen to reduce background binding, including an excess of more than 10-fold purified human Fc.

[0186] For some of the antigens tested, immunotubes were coated overnight at 4°C with 2 mL of antigen (10–30 μg / mL). The volumes of blocking, washing, and elution solutions were increased as needed.

[0187] Amplification of enriched phages The eluted concentrated phage pool was further amplified as follows: Eluted phagemid particles were infected with ER2738 cells at 37°C for 30 minutes. The infected cells were then seeded onto 2x YT agar plates containing 2% glucose, 100 μg / mL ampicillin, and 12.5 μg / mL tetracycline. Colonies were harvested from the plates and 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 phages were purified and quantified using the above process. Typically, ER2738 cells were infected with phages eluted after the final round of panning, and the resulting ER2738 colonies were collected for the supernatant ELISA screening assay.

[0188] Supernatant sandwich Elisa assay A highly sensitive sandwich Elisa assay was constructed to measure the amount of Fab present in the bacterial supernatant. Microplates were coated with polyclonal anti-human IgG (Fab-specific) (Sigma I5260) to capture Fab present in the bacterial supernatant, and then the amount of captured Fab was detected using HRP-labeled goat anti-human Fc. A in each well 450 The Fab binding activity was determined by measuring the ELISA signal. A primary hit was defined as an ELISA signal at least twice the background level, and this was further characterized in the following example (Example 4).

[0189] Using the constructed library, 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) were screened. 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 antibodies that cross-reacted between two species (e.g., bound to TAGT-8H and TAGT-8M). The variant groups of the confirmed binders were a subset of the design variant groups shown in Table 2. The majority of the design variants were also found in the confirmed binders (Table 3). (Refer to the design formulas in Table 2 and the positive hit formulas in Table 3 for comparison). Table 3: Formulas for positively hit HVR-H1 and HVR-H2 design variant sequences TIFF0007856719000008.tif244170TIFF0007856719000009.tif33170

[0190] Example 4: Characterization of antibodies in vitro The Fab corresponding to the primary hit identified in Example 3 above was tagged with a His6 tag at the C-terminus of the CH1 domain, overexpressed in E. coli, and purified using Ni-NTA resin (Thermo Fisher Scientific) according to the manufacturer's instructions. Affinity was measured using the ForteBio Octet RED96 System. Briefly, to capture the antigen Fc-His fusion protein (Sino Biological #10039-H03H), an AHC sensor (anti-human IgG-Fc capture dip and read biosensor) was used and immersed in wells containing purified Fab diluted to 5-10 μg / mL with kinetic buffer (see also ForteBio, Anti-human IgG Capture (AHC) Biosensors, Product Insert 41-0072-PD (2008); Yang et al. (2016), Anal. Biochem. 508:78-96). The acquired ForteBio data was processed using Data Acquisition software 7.1, and the kinetic data was fitted to a 1:1 Langmuir-coupled model. The solubility temperature of Fab was measured by a Differential Scanning Fluorimetry (DSF) assay. Briefly, temperature and fluorescence monitoring were performed using a qPCR instrument (real-time PCR). SYPRO® Orange was diluted 50-fold to 100-fold in PBS buffer from a 5000x stock solution, 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 to 483 nm, and the emission wavelength to 568 nm. The temperature was increased from 25°C to 90°C at an increment of 1.2-1.3°C per minute, with an equilibrium time of 15 seconds at each measurement temperature. The data was analyzed using LightCycler® 480 Software. Tm, the midpoint of the hydrophobic exposure, was defined as the temperature corresponding to the maximum value 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] Twelve 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. Sequence identity between human antigen TAGT-8H and mouse antigen TAGT-8M was 70%, while sequence identity between human antigen TAGT-10H and mouse antigen TAGT-10M was 60%. Multiple high-affinity antibodies targeting 14 different antigens were successfully identified and selected from the dynamic library. Affinity for most binders was in the nanomolar range, but some reached the sub-nanomolar range (Figure 2A). In addition, the identified binders showed good stability, as shown in Figure 2B.

[0192] Example 5: Application of a Dynamic Heavy Chain Library To further investigate the robustness and flexibility of the heavy chain library, 10 7 Libraries were screened for the 14 target antigens described in Example 4 above by pairing heavy chains with various light chain libraries having diversity ranging from ~280 to even a single light chain (i.e., a common light chain). While exploring the flexibility and / or dynamic diversity of the light chains themselves, the limits of diversity design in both heavy chain and light chain libraries were explored based on the physical size of each pairing partner (e.g., 10 7We explored light chain libraries with diversity ranging from ~280, 20, to single light chains by preparing them. The ability of these dynamic light chain libraries in pairing with dynamic heavy chain libraries provided a strong rationale for library design when generating and manipulating diverse antibody hits / reads against known and challenging target antigens. High-affinity positive hits were identified from each library tested, and a total of 690 unique positive hits were measured and confirmed by affinity data (Table 4). We investigated 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 in species cross-reactivity between human and mouse targets with approximately 60% sequence identity). Positive hits were observed using each combination 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 in the construction of antibody and protein libraries that recognize a wide range of targets for therapeutic, diagnostic, and / or research reagents when grafted or designed into antibody (and / or alternative protein) scaffolds. 7 The dynamic properties of these heavy chain hypervariable region units when paired with light chain libraries (ranging from ~280 to a single unique sequence) provide a strong demonstration of the dynamic antibody design concept for creating novel conjugation reagents useful in therapeutic, clinical, and / or research environments. Table 4: Affinity data of confirmed hits TIFF0007856719000010.tif253170TIFF0007856719000011.tif255170TIFF0007856719000012.tif255170TIFF0007856719000013.tif255170TIFF0007856719000014.tif255170TIFF0007856719000015.tif255170TIFF0007856719000016.tif255170TIFF0007856719000017.tif255170TIFF0007856719000018.tif255170TIFF0007856719000019.tif255170TIFF0007856719000020.tif255170TIFF0007856719000021.tif255170TIFF0007856719000022.tif255170TIFF0007856719000023.tif255170TIFF0007856719000024.tif255170TIFF0007856719000025.tif255170TIFF0007856719000026.tif255170TIFF0007856719000027.tif255170TIFF0007856719000028.tif255170TIFF0007856719000029.tif255170TIFF0007856719000030.tif255170TIFF0007856719000031.tif255170TIFF0007856719000032.tif255170TIFF0007856719000033.tif255170TIFF0007856719000034.tif255170TIFF0007856719000035.tif255170TIFF0007856719000036.tif255170TIFF0007856719000037.tif255170TIFF0007856719000038.tif255170TIFF0007856719000039.tif255170TIFF0007856719000040.tif255170TIFF0007856719000041.tif255170TIFF0007856719000042.tif255170TIFF0007856719000043.tif255170TIFF0007856719000044.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 HVR-H2 sequences are paired with different HVR-H3 and VL sequences. For example, hits 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 rates and target binding numbers for HVR-H1 and HVR-H2 identified during library analysis. While we do not wish to be bound by theory, a large number of antigens to which an antibody containing a given hypervariable region binds may indicate a high degree of flexibility in that particular hypervariable region, while a high segment utilization rate of a given hypervariable region may indicate robust folding of the hypervariable region (and surrounding polypeptide sequences). Table 5: Target binding capability of HVR-H1 and HVR-H2 design variants TIFF0007856719000045.tif148170

[0195] Table 6 below shows the sequence usage rate and antigen binding count for HVR-H1 and HVR-H2 combinations identified during library analysis. Table 6: Usage rates of combined design variants for HVR-H1 and HVR-H2 TIFF0007856719000046.tif249170TIFF0007856719000047.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 as appearing in more than one of the unique antibody hits mentioned above. These HVRs, when combined with various HVR-H3 and variable light chain domains, were capable of forming antibodies that bind to multiple antigens. An additional 65 novel HVR-H1 and HVR-H2 sequence combinations were identified as appearing in more than one of the unique antibody hits mentioned above. Table 7 below shows the use of HVR-H1 and HVR-H2 and the number of antigens bound when these novel HVR sequences were analyzed in a library. Table 7: New HVR-H1 and HVR-H2 sequence usage TIFF0007856719000048.tif149170TIFF0007856719000049.tif251170TIFF0007856719000050.tif252170TIFF0007856719000051.tif245170

[0197] Table 8 below shows the use of new HVR-H1 and HVR-H2 sequence combinations and their antigen binding numbers. Table 8: New HVR-H1 and HVR-H2 combination usage TIFF0007856719000052.tif227170TIFF0007856719000053.tif208170

[0198] Table 9 shows affinity data for unique hits using the novel HVR-H1 and HVR-H2 sequences presented. Table 9: Affinity data of hits confirmed using the new HVR-H1 and HVR-H2 sequences TIFF0007856719000054.tif248170TIFF0007856719000055.tif255170TIFF0007856719000056.tif254170TIFF0007856719000057.tif255170TIFF0007856719000058.tif255170TIFF0007856719000059.tif255170TIFF0007856719000060.tif255170TIFF0007856719000061.tif255170TIFF0007856719000062.tif255170TIFF0007856719000063.tif255170TIFF0007856719000064.tif255170TIFF0007856719000065.tif255170TIFF0007856719000066.tif255170TIFF0007856719000067.tif255170TIFF0007856719000068.tif255170TIFF0007856719000069.tif255170TIFF0007856719000070.tif255170TIFF0007856719000071.tif255170TIFF0007856719000072.tif255170TIFF0007856719000073.tif255170TIFF0007856719000074.tif255170TIFF0007856719000075.tif255170TIFF0007856719000076.tif255170TIFF0007856719000077.tif255170TIFF0007856719000078.tif255170TIFF0007856719000079.tif255170TIFF0007856719000080.tif255170TIFF0007856719000081.tif255170TIFF0007856719000082.tif255170TIFF0007856719000083.tif255170TIFF0007856719000084.tif255170TIFF0007856719000085.tif255170TIFF0007856719000086.tif255170TIFF0007856719000087.tif255170TIFF0007856719000088.tif255170TIFF0007856719000089.tif25 5170TIFF0007856719000090.tif255170TIFF0007856719000091.tif255170TIFF0007856719000092.tif255170T IFF0007856719000093.tif255170TIFF0007856719000094.tif255170TIFF0007856719000095.tif255170TIFF00 07856719000096.tif255170TIFF0007856719000097.tif255170TIFF0007856719000098.tif255170TIFF0007856 719000099.tif255170TIFF0007856719000100.tif255170TIFF0007856719000101.tif255170TIFF000785671900 0102.tif255170TIFF0007856719000103.tif255170TIFF0007856719000104.tif255170TIFF0007856719000105. tif255170TIFF0007856719000106.tif255170TIFF0007856719000107.tif255170TIFF0007856719000108.tif25 5170TIFF0007856719000109.tif255170TIFF0007856719000110.tif255170TIFF0007856719000111.tif130170.

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

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

[0201] Exemplary hits IDs 3757 and 5103 contained the same HVR-H1 and HVR-H2 sequences (SEQ ID NOs. 1 and 122) and the same heavy chain variable region, but when combined with different variable light chain domains, they bound to two different target antigens (TAGT-6 and TAGT-10, respectively). Two additional hits with the 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. Several other hits with the same HVR-H1 and HVR-H2 sequences exhibited species cross-reactivity.

[0203] A novel method employing the hypervariable region of antibodies, redefined based on structural and sequence variability, to identify dynamic motifs, V H The components are paired V L A limited number of Vs that can bind to the same or multiple different targets depending on the segment. H The design of components has become possible. The data and antibodies described herein, whether their heavy chain libraries are used as a whole set or a subset, are relevant to antibody discovery. H It demonstrates that it is robust enough to function as a component. array Unless otherwise specified, all polypeptide sequences are written from the N-terminus to the C-terminus. Unless otherwise specified, all polynucleotide sequences are written from 5' to 3'. Designed HVR-H1 sequence 1: FTFTDYGIHWV (Sequence ID 1) Designed HVR-H1 sequence 2:FTFTGYAIHWV (Sequence ID 2) Designed HVR-H1 sequence 3: FTFTNYGIHWV (Sequence ID 3) Designed HVR-H1 sequence 4: YTFSDYAIHWV (Sequence ID 4) Designed HVR-H1 sequence 5: YTFSDYGIHWV (Sequence ID 5) Designed HVR-H1 sequence 6: YTFSGYAIHWV (Sequence ID 6) Designed HVR-H1 sequence 7:YTFSGYGIHWV (sequence number 7) Designed HVR-H1 sequence 8:YTFSNYGIHWV (sequence number 8) Designed HVR-H1 sequence 9:YTFSSYGIHWV (Sequence ID 9) Designed HVR-H1 sequence 10: YTFSGYWIHWV (Sequence ID 10) Designed HVR-H1 sequence 11:YTFSNYWIHWV (sequence number 11) Designed HVR-H1 sequence 12: FTFSGYWIHWV (Sequence ID 12) Designed HVR-H1 sequence 13:FTFSNYWIHWV (Sequence ID 13) Designed HVR-H1 sequence 14:YTFSDYWIHWV (sequence number 14) Designed HVR-H1 sequence 15:YSISSGHHWAWI(sequence number 15) Designed HVR-H1 sequence 16: YSISSGHYWNWI (Sequence ID 16) Designed HVR-H1 sequence 17:YSISSGHYWSWI (sequence number 17) Designed HVR-H1 sequence 18:YSISSGHYWTWI (Sequence ID 18) Designed HVR-H1 sequence 19:YSISSGYHWAWI(Sequence ID 19) Designed HVR-H1 sequence 20: YSISSGYHWDWI (Sequence ID 20) Designed HVR-H1 sequence 21:YSISSGYHWGWI (Sequence ID 21) Designed HVR-H1 sequence 22: YSISSGYHWNWI (Sequence ID 22) Designed HVR-H1 sequence 23:YSISSGYHWSWI (sequence number 23) Designed HVR-H1 sequence 24: YSISSGHHWDWI (Sequence ID 24) Designed HVR-H1 sequence 25:YSISSGYYWDWI (Sequence ID 25) Designed HVR-H1 sequence 26: YSISSGYYWNWI (Sequence ID 26) Designed HVR-H1 sequence 27:YSISSGYYWTWI (sequence number 27) Designed HVR-H1 sequence 28:YSITSGHHWAWI (sequence number 28) Designed HVR-H1 sequence 29:YSITSGHHWDWI (Sequence ID 29) Designed HVR-H1 sequence 30: YSITSGHHWGWI (Sequence ID 30) Designed HVR-H1 sequence 31:YSITSGHHWNWI (Sequence ID 31) Designed HVR-H1 sequence 32:YSITSGHHWSWI (sequence number 32) Designed HVR-H1 sequence 33:YSISSGHHWGWI (Sequence ID 33) Designed HVR-H1 sequence 34:YSITSGHYWAWI (Sequence ID 34) Designed HVR-H1 sequence 35:YSITSGHYWDWI (Sequence ID 35) Designed HVR-H1 sequence 36: YSITSGHYWGWI (Sequence ID 36) Designed HVR-H1 sequence 37:YSITSGHYWNWI (Sequence ID 37) Designed HVR-H1 sequence 38:YSITSGHYWSWI (Sequence ID 38) Designed HVR-H1 sequence 39:YSITSGYHWAWI (Sequence ID 39) Designed HVR-H1 sequence 40: YSITSGYHWGWI (Sequence ID 40) Designed HVR-H1 sequence 41:YSISSGHHWNWI (Sequence ID 41) Designed HVR-H1 sequence 42:YSITSGYHWNWI (Sequence ID 42) Designed HVR-H1 sequence 43:YSITSGYHWSWI (sequence number 43) Designed HVR-H1 sequence 44:YSITSGYYWDWI (Sequence ID 44) Designed HVR-H1 sequence 45:YSISSGHHWTWI (sequence number 45) Designed HVR-H1 sequence 46: YSISSGHYWDWI (Sequence ID 46) Designed HVR-H1 sequence 47:FSLSTSGVAVSWI (Sequence ID 47) Designed HVR-H1 sequence 48: FSLSTGGVAVGWI (Sequence number 48) Designed HVR-H1 sequence 49: FSLSTGGVAVSWI (Sequence ID 49) Designed HVR-H1 sequence 50: FSLSTGGVGVAWI (Sequence number 50) Designed HVR-H1 sequence 51: FSLSTGGVGVSWI (Sequence ID 51) Designed HVR-H1 sequence 52:FSLSTSGVAVAWI (Sequence ID 52) Designed HVR-H1 sequence 53:FTFSDYAIHWV (Sequence ID 137) Designed HVR-H1 sequence 54:FTFSDYGIHWV (Sequence ID 138) Designed HVR-H1 sequence 55:YTFSNYAIHWV (Sequence ID 139) Designed HVR-H1 sequence 56:YTFSSYAIHWV (Sequence ID 140) Designed HVR-H1 sequence 57:YTFTDYAIHWV (Sequence ID 141) Designed HVR-H1 sequence 58:YTFTDYGIHWV (sequence number 142) Designed HVR-H1 sequence 59:YTFTNYAIHWV (Sequence ID 143) Designed HVR-H1 sequence 60:YTFTNYGIHWV (Sequence ID 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: YSSISSGYYWGWI (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: YSSISSGHYWAWI (SEQ ID NO: 153) Designed HVR-H1 sequence 70: YSSISSGHYWGWI (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 (Sequence ID 58) Designed HVR-H2 sequence 7:LARIDWDDDKYYSPSLKSRL (Sequence ID 59) Designed HVR-H2 sequence 8:LARIDWDDDKYYSTSLKSRL (Sequence ID 60) Designed HVR-H2 sequence 9:LARIDWDGDKYYSTSLKSRL (Sequence ID 61) Designed HVR-H2 sequence 10:IGDIYHSGSTYYSPSLKSRV (Sequence ID 62) Designed HVR-H2 sequence 11:IGEIYHSGSTYYSPSLKSRV (Sequence ID 63) Designed HVR-H2 sequence 12:IGEIYYSGSTYYSPSLKSRV (sequence number 64) Designed HVR-H2 sequence 13:IGSIYHSGNTNYNPSLKSRV (Sequence ID 65) Designed HVR-H2 sequence 14:IGEIYHSGNTYYNPSLKSRV (sequence number 66) Designed HVR-H2 sequence 15:IGEIYHSGSTYYNPSLKSRV (sequence number 67) Designed HVR-H2 sequence 16:IGEIYYSGSTYYNPSLKSRV (sequence number 68) Designed HVR-H2 sequence 17:IGDIYHSGNTYYNPSLKSRV (Sequence ID 69) Designed HVR-H2 sequence 18:IGDIYHSGSTYYNPSLKSRV (Sequence ID 70) Designed HVR-H2 sequence 19:VSAISGYGDTTYYADSVKGRF (Sequence ID 71) Designed HVR-H2 sequence 20: VSAISGYGGSTYYADSVKGRF (Sequence ID 72) Designed HVR-H2 sequence 21:VSAISGYGGTTYYADSVKGRF (Sequence ID 73) Designed HVR-H2 sequence 22:VSGISGAGDTTYYADSVKGRF (Sequence ID 74) Designed HVR-H2 sequence 23:VSGISGDGDTTYYADSVKGRF (Sequence ID 75) Designed HVR-H2 sequence 24:VSGISGDGGSTYYADSVKGRF (Sequence ID 76) Designed HVR-H2 sequence 25:VSGISGYGDTTYYADSVKGRF (Sequence ID 77) Designed HVR-H2 sequence 26:VSGISGYGGTTYYADSVKGRF (Sequence ID 78) Designed HVR-H2 sequence 27:VSVISGDGDTTYYADSVKGRF (Sequence ID 79) Designed HVR-H2 sequence 28:VSVISGYGGSTYYADSVKGRF (Sequence ID 80) Designed HVR-H2 sequence 29:VSGISGDGSTTYYADSVKGRF (Sequence ID 81) Designed HVR-H2 sequence 30:VSGISGYGSTTYYADSVKGRF (Sequence ID 82) Designed HVR-H2 sequence 31:VSVISGSGSTTYYADSVKGRF (Sequence ID 83) Designed HVR-H2 sequence 32:VSVISGYGSSTYYADSVKGRF (Sequence ID 84) Designed HVR-H2 sequence 33:VSVISGYGSTTYYADSVKGRF (Sequence ID 85) Designed HVR-H2 sequence 34:VSAISGYGSTTYYADSVKGRF (Sequence ID 86) Designed HVR-H2 sequence 35:VSSISGYGDTTYYADSVKGRF (Sequence ID 87) Designed HVR-H2 sequence 36:VSSISGYGGSTYYADSVKGRF (Sequence ID 88) Designed HVR-H2 sequence 37:VSSISGYGGTTYYADSVKGRF (Sequence ID 89) Designed HVR-H2 sequence 38:VSYISGAGDTTYYADSVKGRF (Sequence ID 90) Designed HVR-H2 sequence 39:VSSISGAGDTTYYADSVKGRF (Sequence ID 91) Designed HVR-H2 sequence 40:VSYISGAGGTTYYADSVKGRF (Sequence ID 92) Designed HVR-H2 sequence 41:VSYISGDGDTTYYADSVKGRF (Sequence ID 93) Designed HVR-H2 sequence 42:VSYISGDGGSTYYADSVKGRF (Sequence ID 94) Designed HVR-H2 sequence 43:VSYISGDGGTTYYADSVKGRF (Sequence ID 95) Designed HVR-H2 sequence 44:VSYISGSGDTTYYADSVKGRF (Sequence ID 96) Designed HVR-H2 sequence 45:VSSISGAGGSTYYADSVKGRF (Sequence ID 97) Designed HVR-H2 sequence 46:VSYISGYGDTTYYADSVKGRF (Sequence ID 98) Designed HVR-H2 sequence 47:VSYISGYGGTTYYADSVKGRF (Sequence ID 99) Designed HVR-H2 sequence 48:VSSISGAGGTTYYADSVKGRF (Sequence ID 100) Designed HVR-H2 sequence 49:VSSISGDGDTTYYADSVKGRF (Sequence ID 101) Designed HVR-H2 sequence 50:VSSISGDGGTTYYADSVKGRF (Sequence ID 102) Designed HVR-H2 sequence 51:VSSISGAGSSTYYADSVKGRF (Sequence ID 103) Designed HVR-H2 sequence 52:VSSISGAGSTTYYADSVKGRF (Sequence ID 104) Designed HVR-H2 sequence 53:VSSISGDGSSTYYADSVKGRF (Sequence ID 105) Designed HVR-H2 sequence 54:VSSISGDGSTTYYADSVKGRF (Sequence ID 106) Designed HVR-H2 sequence 55:VSSISGYGSSTYYADSVKGRF (Sequence ID 107) Designed HVR-H2 sequence 56:VSSISGYGSTTYYADSVKGRF (Sequence ID 108) Designed HVR-H2 sequence 57:IGWINPNRGDTKYAQKFQGRV (Sequence ID 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: IGRINPNFGDTNYAQKFQGRV (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 (Sequence ID 127) Designed HVR-H2 sequence 76:IGWISPSGGSTNYAQKFQGRV (Sequence ID 128) Designed HVR-H2 sequence 77:IGWISPSSGSTKYAQKFQGRV (Sequence ID 129) Designed HVR-H2 sequence 78:IGWISPSSGSTNYAQKFQGRV (Sequence ID 130) Designed HVR-H2 sequence 79:IGWISPSGGSTKYAQKFQGRV (Sequence ID 131) Designed HVR-H2 sequence 80:IGIIYPSGGGTNYAQKFQGRV (Sequence ID 132) Designed HVR-H2 sequence 81:IGIISPSGGGTKYAQKFQGRV (Sequence ID 133) Designed HVR-H2 sequence 82:IGIISPSGGGTNYAQKFQGRV (Sequence ID 134) Designed HVR-H2 sequence 83:IGIIYPSGGSTNYAQKFQGRV (Sequence ID 135) Designed HVR-H2 sequence 84:VGRIKSKTDGYTTEYAAPVKGRF (Sequence ID 136) Designed HVR-H2 sequence 85:VSAISGSGSTTYYADSVKGRF (Sequence ID 159) Designed HVR-H2 sequence 86:VSSISGSGDTTYYADSVKGRF (Sequence ID 160) Designed HVR-H2 sequence 87:VSSISGSGGSTYYADSVKGRF (Sequence ID 161) Designed HVR-H2 sequence 88:VSSISGSGGTTYYADSVKGRF (Sequence ID 162) Designed HVR-H2 sequence 89:VSSISGDGGSTYYADSVKGRF (Sequence ID 163) Designed HVR-H2 sequence 90:VSSISGSGSTTYYADSVKGRF (Sequence ID 164) Framework FW-H1 array: EVQLVESGGGLVQPGGSLRLSCAASG (Sequence ID 165) Framework FW-H2 array: RQAPGKGLEW (sequence number 166) Framework FW-H3 sequence: TISSRDNSKNTLYLQLNSLRAEDTAVYYC (Sequence ID 167) Framework FW-H4 array: WGQGTLVTVSS (Sequence ID 168) Hit ID 4029-VH EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYHWGWIRQAPGKGLEWVSYISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDYGDYYGFDYWGQGTLVTVSS(Sequence ID 169) Hit ID 4029-VL DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYRTPFTFGQGTKVEIKR(Sequence ID 170) Hit ID 7097-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHHWDWIRQAPGKGLEWVSYISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGSDAVLGDWFAYWGQGTLVTVSS(Sequence ID 171) Hit ID 7097-VL DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGTKVEIKR(Sequence ID 172) Hit ID 5906-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWNWIRQAPGKGLEWVSYISGDGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLGGYYGWGRYFDYWGQGTLVTVSS(Sequence ID 173) Hit ID 5906-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR(Sequence ID 174) Hit ID 7040-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYYWNWIRQAPGKGLEWIGWISPSGGSTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDLTAGGFDYWGQGTLVTVSS(Sequence ID 175) Hit ID 7040-VL DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPLTFGQGTKVEIKR(Sequence ID 176) Hit ID 5924-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWGWIRQAPGKGLEWIGIISPSSGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGAGVHYALDYWGQGTLVTVSS(Sequence ID 177) Hit ID 5924-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR(Sequence ID 178) Hit ID 4034-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWVSSISGYGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARERYYGSTDYAFDYWGQGTLVTVSS(Sequence ID 179) Hit ID 4034-VL DIQLTQSPSSLSASVGDRVTITCSASSRVSHVFWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCLQGTHFPWTFGQGTKVEIKR (Sequence ID 180) Hit ID 6010-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWIGWINPNRGDTNYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARDYYGDFDYWGQGTLVTVSS(Sequence ID 181) Hit ID 6010-VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKVEIKR (Sequence ID 182) Hit ID 7183-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWNWIRQAPGKGLEWVSSISGYGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAREGSDTVLGDWFAYWGQGTLVTVSS(Sequence ID 183) Hit ID 7183-VL DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKR(Sequence ID 184) Hit ID 4036-VH EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGVGVGWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARERYGSYYFDYWGQGTLVTVSS(Sequence ID 185) Hit ID 4036-VL DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGKSFLDWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYRIPPTFGQGTKVEIKR (Sequence ID 186) Hit ID 5115-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGHYWGWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARESYYAFDYWGQGTLVTVSS(Sequence ID 187) Hit ID 5115-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTTPLTFGQGTKVEIKR (Sequence ID 188) Hit ID 5404-VH EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWAWIRQAPGKGLEWIGEIYHSGSTYYSPSLKSRVTISR DNSKNTLYLQLNSLRAEDTAVYYCARSPYYYGVFDYWGQGTLVTVSS (Sequence ID 189) Hit ID 5404-VL DIQLTQSPSSLSASVGDRVTITCSASSRVGSVYWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYTHDPVTFGQGTKVEIKR (Sequence ID 190) Hit ID 3757-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGVGDFDYWGQGTLVTVSS(Sequence ID 191) Hit ID 3757-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR(Sequence ID 192) Hit ID 5103-VH EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYGIHWVRQAPGKGLEWIGWISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHSYYGVGDFDYWGQGTLVTVSS(Sequence ID 193) Hit ID 5103-VL DIQLTQSPSSLSASVGDRVTITCRASQSVSSYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIKR(Sequence ID 194) Hit ID 4027-VH EVQLVESGGGLVQPGGSLRLSCAASGYSITSGHHWNWIRQAPGKGLEWIGWISPSSGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARGFDGFHYWGQGTLVTVSS(Sequence ID 195) Hit ID 4027-VL DIQLTQSPSSLSASVGDRVTITCRASESVDFYGISFLPWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSWPWTFGQGTKVEIKR(Sequence ID 196) VH in Figure 1B EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYGIHWVRQAPGKGLEWVSGISGAGDTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARERDYDFDYWGQGTLVTVSS(Sequence ID 197) Equation (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) (Sequence ID 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) (Sequence ID 199) Formula (III) FSLSTX1GVX2VX3WI (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) (Sequence ID 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) (Sequence ID 201) Formula (V) IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y) (Sequence ID 202) Equation (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) (Sequence ID 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) (Sequence ID 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) (Sequence ID 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) (Sequence ID 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) (Sequence ID 207) Formula (XI) IGX1IX2X3SGSTYYSPSLKSRV (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y) (Sequence ID 208) Equation (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) (Sequence ID 209) Equation (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) (Sequence ID 210) Primer F_1999 CGTTTGTCCTGTGCAGCTTCCGG (Sequence ID 211) Primer R_1999 CGAGGCCCTTACCCGGGGCCTGACG (Sequence ID 212) Primer F_2003 CCGGGTAAGGGCCTCGAGTGG (Sequence No. 213) Primer R_2003 GAGCACGTCCGTTCGAATTGTCGCGACTTATAG (Sequence ID 214) Primer S1089 ACAACTGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTG (Sequence No. 215) Primer S1090 GAGGAGACGGTGACTAGTGTTCCTTGACCCCA (Sequence ID 216) Primer F_2898 TACTTATGTAGGCGATCGGGTCACCATCACCTGC (Sequence ID 217) Primer R_2898 CGGAGCTTTTCCTGGTTTCTGTTGATAC (Sequence ID 218) Primer F_2013 GAAACCAGGAAAAGCTCCGAAG (Sequence No. 219) Primer R_2013 CGTCCCGGAACCGGATCCAGAGAAGCGAG (Sequence No. 220) Primer F2929 ACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAAC (Sequence ID 221) Primer R2929 GATCTCCACCTTGGTACCCTGTCCGAA (Sequence ID 222) HVR-H3 sequence 1: ARDLGGYYGWGRYFDY (sequence number 223) HVR-H3 sequence 2: ARDLTAGGFDY (sequence number 224) HVR-H3 sequence 3: ARDPGVGGFDV (Sequence ID 225) HVR-H3 sequence 4: ARDPGYTWYFDV (sequence number 226) HVR-H3 sequence 5: ARDYGDYYGFDY (sequence number 227) HVR-H3 sequence 6: ARDYGYTWYFDV (sequence number 228) HVR-H3 sequence 7: ARDYYGDFDY (Sequence ID 229) HVR-H3 sequence 8: AREGSDAVLGDWFAY (Sequence ID 230) HVR-H3 sequence 9: AREGSDTVLGDWFAY (Sequence ID 231) HVR-H3 sequence 10:ARERYGSYYFDY (Sequence ID 232) HVR-H3 sequence 11:ARERYYGSTDYAFDY (Sequence ID 233) HVR-H3 sequence 12: ARESYYAFDY (sequence number 234) HVR-H3 sequence 13:ARGAGVHYALDY (Sequence ID 235) HVR-H3 sequence 14:ARGFDGFHY (sequence number 236) HVR-H3 sequence 15: ARGFYGGALDV (Sequence ID 237) HVR-H3 sequence 16:ARGGGGYYFDV (sequence number 238) HVR-H3 sequence 17:ARGGGLGFDY (sequence number 239) HVR-H3 sequence 18:ARGGLGPFDI (sequence number 240) HVR-H3 sequence 19:ARGGSDTVIGDWFAY (sequence number 241) HVR-H3 sequence 20: ARGGVGPFDI (sequence number 242) HVR-H3 sequence 21:ARGGYGGYLDV (sequence number 243) HVR-H3 sequence 22:ARGLSSGYFDY (Sequence ID 244) HVR-H3 sequence 23:ARGSWYFDV (sequence number 245) HVR-H3 sequence 24:ARGTRGLDY (sequence number 246) HVR-H3 sequence 25:ARGYSDYFDY (sequence number 247) HVR-H3 sequence 26:ARGYYYGRAFDY (sequence number 248) HVR-H3 sequence 27:ARHSYYGVGDFDY (Sequence ID 249) HVR-H3 sequence 28: ARLFEGFPY (Sequence ID 250) HVR-H3 sequence 29:ARLYDYFAY (sequence number 251) HVR-H3 sequence 30:ARSGYYALDY (sequence number 252) HVR-H3 sequence 31:ARSPYYYGVFDY (sequence number 253) HVR-H3 sequence 32:ARSYVYFDY (sequence number 254) HVR-H3 sequence 33:ARDGLGLRGVYYYYYGLDV (Sequence ID 255) HVR-H3 sequence 34:ARVGESGGIESPYYYYGLDV (sequence number 256) HVR-L1 sequence 1:RASESVDFYGISFLP (Sequence ID 257) HVR-L1 sequence 2:RASQSVDFYGISFLA (sequence number 258) HVR-L1 sequence 3:RASQSVDFYGKSFLD (Sequence ID 259) HVR-L1 sequence 4:SASSRVGSVY (Sequence ID 260) HVR-L1 sequence 5:SASSRVSHVF (Sequence ID 261) HVR-L1 sequence 6:RASQGISSYLA (Sequence ID 262) HVR-L1 sequence 7:RASQSVSSYLA (sequence number 263) HVR-L1 sequence 8:RASQSISSYLN (Sequence ID 264) HVR-L3 sequence 1:FCLQGTHFPWT (Sequence ID 265) HVR-L3 sequence 2: YCQQSYRTPFT (Sequence ID 266) HVR-L3 Array 3: YCQQSYSWPWT (Sequence ID 267) HVR-L3 sequence 4:YCQQYTHDPVT (Sequence ID 268) HVR-L3 sequence 5:YCQQYYRIPPT (Sequence ID 269) HVR-L3 sequence 6: YCQHHYGTPLT (Sequence ID 270) HVR-L3 sequence 7:YCQQSYSTPLT (Sequence ID 271) HVR-L3 sequence 8:YCQQSYSTPPT (Sequence ID 272) HVR-L3 sequence 9:YCQQYYSTPLT (Sequence ID 273) HVR-L3 sequence 10:YCQQYYTTPLT (Sequence ID 274) <Further Embodiments of the Invention> [Embodiment 1] A library comprising polynucleotides, wherein one of the polynucleotides encodes an antibody heavy chain variable region comprising HVR-H1, HVR-H2, and HVR-H3. The aforementioned HVR-H1 is, (Formula I)X1TFX2X3YX4IHWV(Sequence ID 198)(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); (Formula II)YSIX1SGX2X3WX4WI(Sequence ID 199)(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); and (Formula III)FSLSTX1GVX2VX3WI (Sequence No. 200) (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned HVR-H2 is, (Formula IV)LAX1IX2WX3X4DKX5YSX6SLKSRL(Sequence ID 201)(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); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV(Sequence ID 202)(wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y); (Formula VI)IGX1IYX2SGX3TX4YNPSLKSRV(Sequence ID 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)VSX1ISGX2GX3X4TYYADSVKGRF(Sequence ID 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(Sequence ID 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(Sequence ID 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 (Sequence 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) The amino acid sequence includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned library. [Embodiment 2] At least two, at least three, at least four, at least five, or at least ten of the polynucleotides encode a heavy chain variable region including HVR-H1, HVR-H2, and HVR-H3. The aforementioned HVR-H1 is, (Formula I)X1TFX2X3YX4IHWV(Sequence ID 198)(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); (Formula II)YSIX1SGX2X3WX4WI(Sequence ID 199)(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); and (Formula III)FSLSTX1GVX2VX3WI (Sequence No. 200) (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned HVR-H2 is, (Formula IV)LAX1IX2WX3X4DKX5YSX6SLKSRL(Sequence ID 201)(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); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV(Sequence ID 202)(wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y); (Formula VI)IGX1IYX2SGX3TX4YNPSLKSRV(Sequence ID 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)VSX1ISGX2GX3X4TYYADSVKGRF(Sequence ID 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(Sequence ID 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(Sequence ID 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 (Sequence 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) The amino acid sequence includes an amino acid sequence that follows a formula selected from the group consisting of the following: The library described in Embodiment 1. [Embodiment 3] The aforementioned HVR-H2, (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (Sequence ID 208) (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y); (Formula XII)IGX1IYX2SGX3TX4YNPSLKSRV(Sequence ID 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 (Sequence 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) The amino acid sequence includes an amino acid sequence that follows a formula selected from the group consisting of the following: A library according to Embodiment 1 or 2. [Embodiment 4] The library according to any one of Embodiments 1 to 3, wherein each of the polynucleotides codes for a heavy chain comprising HVR-H1, HVR-H2, and HVR-H3, and HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158. [Embodiment 5] The library according to any one of Embodiments 1 to 4, wherein each of the polynucleotides codes for a heavy chain comprising HVR-H1, HVR-H2, and HVR-H3, and HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52. [Embodiment 6] The library according to any one of Embodiments 1 to 5, wherein each of the polynucleotides codes for a heavy chain comprising HVR-H1, HVR-H2, and HVR-H3, and the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 136 and 159 to 164. [Embodiment 7] The library according to any one of Embodiments 1 to 6, wherein each of the polynucleotides codes for a heavy chain comprising HVR-H1, HVR-H2, and HVR-H3, and the HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 136. [Embodiment 8] The library according to any one of Embodiments 1 to 7, wherein the polynucleotide comprises a unique combination of HVR-H1 and HVR-H2 sequences less than approximately 6.5*10⁴. [Embodiment 9] The library according to Embodiment 8, wherein the polynucleotides include fewer than approximately 6700 unique combinations of HVR-H1 and HVR-H2 sequences. [Embodiment 10] The library according to Embodiment 9, wherein the polynucleotides include a unique combination of approximately 6660 or fewer HVR-H1 sequences and HVR-H2 sequences. [Embodiment 11] The library according to any one of Embodiments 1 to 10, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and the HVR-H2 of the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. [Embodiment 12] The library according to any one of Embodiments 1 to 11, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and the HVR-H2 of the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. [Embodiment 13] The heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); and HVR-H1 includes the amino acid sequence of formula (II) and the amino acid sequence of formula (VI) A library according to Embodiment 1, selected from the group consisting of HVR-H2 containing a sequence; HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII). [Embodiment 14] The heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of SEQ ID NO: 157 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; HVR-H1 includes the amino acid sequence of SEQ ID NO: 1 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 122; HVR-H1 includes the amino acid sequence of SEQ ID NO: 138 and HVR-H2 includes the amino acid sequence of SEQ ID NO: 63; HVR-H1 includes the amino acid sequence of SEQ ID NO: 154 and HVR-H3 includes the amino acid sequence of SEQ ID NO: 63 VR-H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 161; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 145 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 128; HVR-H1 containing the amino acid sequence of SEQ ID NO: 22 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 61; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63 H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 153 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63; HVR-H1 containing the amino acid sequence of SEQ ID NO: 155 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 67; HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 100; HVR-H1 containing the amino acid sequence of SEQ ID NO: 51 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 123 H2; HVR-H1 containing the amino acid sequence of SEQ ID NO: 139 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110; HVR-H1 containing the amino acid sequence of SEQ ID NO: 8 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 126; HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 129; HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 124; HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 130;A library according to Embodiment 1, selected from the group consisting of HVR-H1 containing the amino acid sequence of SEQ ID NO: 150 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 132; HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162; HVR-H1 containing the amino acid sequence of SEQ ID NO: 12 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 82; HVR-H1 containing the amino acid sequence of SEQ ID NO: 149 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 117; and HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 134. [Embodiment 15] The library according to any one of Embodiments 1 to 14, wherein the heavy chain variable region includes an HVR-H3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 223 to 256. [Embodiment 16] The library according to any one of Embodiments 1 to 15, wherein the heavy chain variable region includes FW-H1 containing the amino acid sequence of SEQ ID NO: 165, FW-H2 containing the amino acid sequence of SEQ ID NO: 166, FW-H3 containing the amino acid sequence of SEQ ID NO: 167, and / or FW-H4 containing the amino acid sequence of SEQ ID NO: 168. [Embodiment 17] The library according to Embodiment 1, wherein the heavy chain variable region includes sequences selected from the group consisting of SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, and 195. [Embodiment 18] The library according to any one of embodiments 1 to 17, wherein the polynucleotide encodes a full-length antibody heavy chain. [Embodiment 19] A library according to any one of Embodiments 1 to 18, further comprising a polynucleotide encoding a variable region of an antibody light chain. [Embodiment 20] The library according to Embodiment 19, wherein 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 to 264, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 265 to 274. [Embodiment 21] The library according to Embodiment 19 or 20, wherein the polynucleotide encoding the antibody light chain variable region includes at least one unique sequence. [Embodiment 22] The library according to any one of embodiments 19 to 21, wherein the polynucleotide encoding the antibody light chain variable region comprises at least about 280 unique sequences. [Embodiment 23] The library according to any one of embodiments 19 to 22, wherein the polynucleotide encoding the antibody light chain variable region comprises at least about 105 unique sequences. [Embodiment 24] A library comprising polynucleotides encoding multiple unique antibodies, each antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region of each of the multiple antibodies comprising the same sequence, comprising HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 is (Formula I)X1TFX2X3YX4IHWV(Sequence ID 198)(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); (Formula II)YSIX1SGX2X3WX4WI(Sequence ID 199)(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); and (Formula III)FSLSTX1GVX2VX3WI (Sequence No. 200) (wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned HVR-H2 is, (Formula IV)LAX1IX2WX3X4DKX5YSX6SLKSRL(Sequence ID 201)(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); (Formula V)IGX1IX2X3SGSTYYSPSLKSRV(Sequence ID 202)(wherein X1 is A, D, E, S, or Y, X2 is S or Y, and X3 is H or Y); (Formula VI)IGX1IYX2SGX3TX4YNPSLKSRV(Sequence ID 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)VSX1ISGX2GX3X4TYYADSVKGRF(Sequence ID 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(Sequence ID 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(Sequence ID 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 (Sequence 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) The amino acid sequence includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned library. [Embodiment 25] The aforementioned HVR-H2, (Formula XI)IGX1IX2X3SGSTYYSPSLKSRV (Sequence ID 208) (wherein X1 is A, D, or E, X2 is S or Y, and X3 is H or Y); (Formula XII)IGX1IYX2SGX3TX4YNPSLKSRV(Sequence ID 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 (Sequence 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) The amino acid sequence includes an amino acid sequence that follows a formula selected from the group consisting of the following: The library described in Embodiment 24. [Embodiment 26] The library according to Embodiment 24 or 25, wherein 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. [Embodiment 27] The library according to any one of embodiments 24 to 26, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52. [Embodiment 28] The library according to any one of embodiments 24 to 27, wherein the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 136 and 159 to 164. [Embodiment 29] The library according to any one of embodiments 24 to 28, wherein the heavy chain variable region includes HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 53 to 136. [Embodiment 30] The library according to any one of embodiments 24 to 29, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52 and 137 to 158, and HVR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136 and 159 to 164. [Embodiment 31] The library according to any one of Embodiments 24 to 30, wherein the heavy chain variable region comprises HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 52, and the HVR-H2 of the antibody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 136. [Embodiment 32] The heavy chain variable region comprises three HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 and HVR-H2 include the amino acid sequence of formula (II) and HVR-H2 include the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (VII); HVR-H1 includes the amino acid sequence of formula (I) and HVR-H2 includes the amino acid sequence of formula (IX); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (IV); HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (V); and HVR-H1 includes the amino acid sequence of formula (II) and HVR-H2 includes the amino acid sequence of formula (VI). A library according to Embodiment 24, selected from the group consisting of HVR-H2 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII); HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII); HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V); HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (V); and HVR-H1 c...

Claims

1. A library containing polynucleotide members, all of which encode an antibody heavy chain variable region consisting of three hypervariable regions (HVR) and four framework (FW) regions arranged in the order (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4), All polynucleotide members encode the antibody heavy chain variable region, including HVR-H1 and HVR-H2 as described below. The aforementioned HVR-H1 is, (Formula I) １ TFX ２ X ３ YX ４ IHWV (Sequence ID 198) (wherein X １ is F or Y, and X ２ is S or T, X ３ is D, G, N, or S, 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, X ４ is A, D, G, N, S, or T); and (Formula III) FSLSTX １ GVX ２ VX ３ WI (Sequence ID 200) (where X １ is G or S, X ２ is A or G, and X ３ (It is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The aforementioned HVR-H2 is, (Formula IV) LAX １ IX ２ WX ３ X ４ DKX ５ YSX ６ SLKSRL (Sequence No. 201) (wherein X １ is L or R, X ２ is D or Y, X ３ is A, D, S, or Y, and X ４ is D or G, X ５ is R, S, or Y, and X ６ (is P or T); (Formula V) IGX １ IX ２ X ３ SGSTYYSPSLKSRV (Sequence No. 202) (wherein X １ is A, D, E, S, or Y, and X ２ is S or Y, and X ３ (is H or Y); (Formula VI) IGX １ IYX ２ SGX ３ TX ４ YNPSLKSRV (Sequence No. 203) (wherein X １ is D, E, R, S, or Y, and X ２ is H or Y, X ３ is N or S, X ４ (is N or Y); (Formula VII) VSX １ ISGX ２ GX ３ X ４ TYYADSVKGRF (Sequence No. 204) (wherein X １ is A, G, S, V, or Y, and X ２ is A, D, S, or Y, and X ３ is D, G, or S, X ４ (is S or T); (Formula VIII) IGX １ INPNX ２ GX ３ TX ４ YAQKFQGRV (Sequence ID 205) (wherein X １ is I, R, or W, and X ２ is F or R, X ３ is D, G, or S, X ４ (is K or N); (Formula IX) IGX １ IX ２ PSX ３ GX ４ TX ５ YAQKFQGRV (Sequence No. 206) (wherein X １ is I, R, or W, and X ２ is S or Y, and X ３ is G or S, X ４ is D, G, or S, X ５ (is K or N); and (Formula X) VGRIX １ SKX ２ X ３ GX ４ TTX ５ YAAX ６ VKGRF (Sequence 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, and X ５ is D or E, X ６ (is P or S) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The heavy chain variable region includes FW-H1 containing the amino acid sequence of SEQ ID NO: 165, FW-H2 containing the amino acid sequence of SEQ ID NO: 166, FW-H3 containing the amino acid sequence of SEQ ID NO: 167, and FW-H4 containing the amino acid sequence of SEQ ID NO:

168. The library further comprises at least one polynucleotide encoding the antibody light chain variable region. Library.

2. The aforementioned polynucleotide is 6.5 ＊ 10 ４ The library according to claim 1, comprising unique combinations of HVR-H1 and HVR-H2 sequences less than 1.

3. a. The polynucleotide contains a unique combination of HVR-H1 and HVR-H2 sequences of less than 6700, or b. The library according to claim 2, wherein the polynucleotide comprises a unique combination of HVR-H1 sequences and HVR-H2 sequences of 6660 or less.

4. a. The heavy chain variable region includes HVR-H1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-52 and 137-158, and / or b. The library according to any one of claims 1 to 3, wherein the heavy chain variable region comprises HVR-H2 having 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 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (IX), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (IX), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (IV), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (V), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VI), formula (I HVR-H1 containing the amino acid sequence of formula (VI) and HVR-H2 containing the amino acid sequence of formula (VI), HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VI), HVR-H1 containing the amino acid sequence of formula (III) and HVR-H2 containing the amino acid sequence of formula (VII), HVR-H1 containing the amino acid sequence of formula (II) and HVR-H2 containing the amino acid sequence of formula (VIII), HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (V), and HVR-H1 containing the amino acid sequence of formula (I) and HVR-H2 containing the amino acid sequence of formula (VIII), A library according to claim 1, selected from the group consisting of the following.

6. HVR-H1 and HVR-H2 are, HVR-H1 containing the amino acid sequence of SEQ ID NO: 157 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 1 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 122, HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 154 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 161, HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 145 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 128, HVR-H1 containing the amino acid sequence of SEQ ID NO: 22 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 61, HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, HVR-H1 containing the amino acid sequence of SEQ ID NO: 153 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 63, sequence HVR-H1 containing the amino acid sequence of number 155 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 67, HVR-H1 containing the amino acid sequence of SEQ ID NO: 156 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 100, HVR-H1 containing the amino acid sequence of SEQ ID NO: 51 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162, HVR-H1 containing the amino acid sequence of SEQ ID NO: 138 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 123, HVR-H1 containing the amino acid sequence of SEQ ID NO: 139 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 110 , HVR-H1 containing the amino acid sequence of SEQ ID NO: 8 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 126, HVR-H1 containing the amino acid sequence of SEQ ID NO: 13 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 129, HVR-H1 containing the amino acid sequence of SEQ ID NO: 31 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 124, HVR-H1 containing the amino acid sequence of SEQ ID NO: 25 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 130, HVR-H1 containing the amino acid sequence of SEQ ID NO: 150 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 132,HVR-H1 containing the amino acid sequence of SEQ ID NO: 158 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 162, HVR-H1 containing the amino acid sequence of SEQ ID NO: 12 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 82, HVR-H1 containing the amino acid sequence of SEQ ID NO: 149 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 117, and HVR-H1 containing the amino acid sequence of SEQ ID NO: 7 and HVR-H2 containing the amino acid sequence of SEQ ID NO: 134, A library according to claim 1, selected from the group consisting of the following.

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

8. The library according to claim 1, wherein the heavy chain variable region includes sequences 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 according to any one of claims 1 to 8, wherein the polynucleotides encode full-length antibody heavy chains.

10. a. 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 to 264, and / or HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 265 to 274; and / or b. The polynucleotide encoding the antibody light chain variable region comprises at least one unique sequence, at least 280 unique sequences, or at least 10 ５ Includes a unique array of The library according to any one of claims 1 to 9.

11. When at least one of the HVR-H1 and HVR-H2 in the antibody heavy chain variable region is evaluated by structural determination and / or computer modeling, it takes on multiple three-dimensional structures. The library according to any one of claims 1 to 10.

12. The library according to any one of claims 1 to 11, wherein at least one polynucleotide encoding an antibody heavy chain variable region is present in the vector.

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

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

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

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

17. The library according to claim 16, wherein the heavy chain variable regions of each of the multiple antibodies contain the same sequence.

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

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

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

21. The non-human animal according to claim 20, wherein the non-human animal is a mammal.

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

23. The library according to claim 22, wherein the library comprises phages, and each antigen-binding domain is presented 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 antibody light chain variable regions, a. Screening a library for a first antigen-binding domain that binds to a first antigen, wherein the first antigen-binding domain comprises a first antibody heavy chain variable region and a first antibody light chain variable region, and the library comprises antigen-binding domain members. All antigen-binding domain members include antibody heavy chain variable regions and antibody light chain variable regions, and all of the antibody heavy chain variable regions consist of three hypervariable regions (HVR) and four framework (FW) regions arranged in the order (FW-H1)-(HVR-H1)-(FW-H2)-(HVR-H2)-(FW-H3)-(HVR-H3)-(FW-H4), and all antibody heavy chain variable regions include the following HVR-H1 and HVR-H2, HVR-H1 is, (Formula I) X １ TF X ２ X ３ Y X ４ IHW V (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, X ４ is A, G, or W); (Formula II) YSIX １ SGX ２ X ３ WX ４ WI (Sequence No. 199) (where X １ is S or T, X ２ is H or Y, X ３ is H or Y, X ４ (is A, D, G, N, S, or T); and (Formula III) FSLSTX １ GVX ２ VX ３ WI (Sequence ID 200) (where X １ is G or S, X ２ is A or G, and X ３ (It is A, G, S, or T) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: HVR-H2, (Formula IV) LAX １ IX ２ WX ３ X ４ DKX ５ YSX ６ SLKSRL (Sequence No. 201) (wherein X １ is L or R, X ２ is D or Y, X ３ is A, D, S, or Y, and X ４ is D or G, X ５ is R, S, or Y, and X ６ (is P or T); (Formula V) IGX １ IX ２ X ３ SGSTYYSPSLKSRV (Sequence No. 202) (wherein X １ is A, D, E, S, or Y, and X ２ is S or Y, and X ３ (is H or Y); (Formula VI) IGX １ IYX ２ SGX ３ TX ４ YNPSLKSRV (Sequence No. 203) (wherein X １ is D, E, R, S, or Y, and X ２ is H or Y, X ３ is N or S, X ４ (is N or Y); (Formula VII) VSX １ ISGX ２ GX ３ X ４ TYYADSVKGRF (Sequence No. 204) (wherein X １ is A, G, S, V, or Y, and X ２ is A, D, S, or Y, and X ３ is D, G, or S, X ４ (is S or T); (Formula VIII) IGX １ INPNX ２ GX ３ TX ４ YAQKFQGRV (Sequence ID 205) (wherein X １ is I, R, or W, and X ２ is F or R, X ３ is D, G, or S, X ４ (is K or N); (Formula IX) IGX １ IX ２ PSX ３ GX ４ TX ５ YAQKFQGRV (Sequence No. 206) (wherein X １ is I, R, or W, and X ２ is S or Y, and X ３ is G or S, X ４ is D, G, or S, X ５ (is K or N); and (Formula X) VGRIX １ SKX ２ X ３ GX ４ TTX ５ YAAX ６ VKGRF (Sequence 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, and X ５ is D or E, X ６ (is P or S) It includes an amino acid sequence that follows a formula selected from the group consisting of the following: The antibody heavy chain variable region includes FW-H1 containing the amino acid sequence of SEQ ID NO: 165, FW-H2 containing the amino acid sequence of SEQ ID NO: 166, FW-H3 containing the amino acid sequence of SEQ ID NO: 167, and FW-H4 containing the amino acid sequence of SEQ ID NO:

168. The aforementioned screening, b. Screening the library for a second antigen-binding domain that binds to a second antigen, wherein the library is the same library as the library for a first antigen-binding domain that binds to a first antigen, the second antigen-binding domain includes a second antibody heavy chain variable region and a second antibody light chain variable region, and the second antibody heavy chain variable region has the same sequence as the first antibody heavy chain variable region, and the screening is performed accordingly. c. Producing a bispecific antibody comprising the first antigen-binding domain and the second antigen-binding domain, Generation method.

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