Radically diverse human antibody libraries

Abstract

The present application relates to a radically diverse library of human antibodies. Disclosed are antibody libraries comprising a plurality of antibodies having a combination of non-naturally occurring complementarity determining regions from naturally occurring memory and initial B cells in humans, and wherein the antibody libraries comprise a large number of functional and non-redundant antibodies. Also disclosed are methods of making libraries of antibodies with high levels of functional diversity.

Description

[0001] This application is a divisional application of an application filed on December 18, 2018, with application number 201880089697.0 and invention name “Fundamentally Diverse Human Antibody Library”. Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 607,199, filed December 18, 2017, and U.S. Provisional Application No. 62 / 753,754, filed October 31, 2018, the entire contents of which are incorporated herein by reference. Background of the Invention

[0003] Monoclonal antibodies (mAbs) can be used as therapeutic agents, research tools, and in diagnostic methods, but finding antibodies with affinity for the desired target can be challenging. Antibody libraries provide an effective tool for screening large numbers of antibodies against target compounds. Such libraries are typically based on the rearrangement of naturally occurring variable genes or the introduction of synthetic diversity into antibody sequences. However, natural antibody libraries often have extremely limited diversity, and synthetic libraries can be plagued by non-functional sequences. Therefore, there is a need to develop antibody libraries with a high degree of functional diversity. Summary of the invention

[0004] Provided herein is an antibody library comprising a variety of antibodies. A variety of antibodies may include a VH domain including a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence and a VL domain including a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence. At least one of the VH-CDR3 sequence and the VL-CDR3 sequence may be derived from an initial (naïve) B cell. In some embodiments, if only one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from an initial B cell, the VH-CDR3 sequence or the VL-CDR3 sequence that is not derived from an initial B cell is derived from a memory cell. The VH-CDR1 sequence, the VH-CDR2 sequence, the VL-CDR1 sequence, and the VL-CDR2 sequence may be derived from a memory B cell. In some embodiments, at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from an initial B cell is a naturally occurring sequence. In some embodiments, the VH-CDR3 sequence or VL-CDR3 sequence derived from memory cells is a naturally occurring sequence. In some embodiments, the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells are naturally occurring sequences. In some embodiments, at least one of the VH-CDR3 sequence and VL-CDR3 sequence derived from initial B cells contains at least 80% sequence homology with a naturally occurring sequence. In some embodiments, the VH-CDR3 sequence or VL-CDR3 sequence derived from memory cells contains at least 80% sequence homology with a naturally occurring sequence. In some embodiments, the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells contain at least 80% sequence homology with a naturally occurring sequence. In some embodiments, the VL domain is a VK domain or a Vλ domain. In some embodiments, the initial B cell is a CD27- / IgM+ B cell or a CD27- / IgD+ B cell. In some embodiments, the memory B cell is selected from CD27+ / IgG+ B cells, CD27+ / IgM+ B cells, IgA+ B cells and combinations thereof. In some embodiments, the initial B cells and memory B cells are from samples including multiple initial B cells and memory B cells sampled from multiple individuals. In some embodiments, multiple individuals are at least 50 individuals. In some embodiments, multiple antibodies are expressed on the surface of multiple phages. In some embodiments, multiple phages are bacterial phages or phagemids. In some embodiments, each phage in a plurality of phages comprises a nucleic acid sequence encoding the following: i) antibodies in a plurality of antibodies, and ii) genes encoding phage coat proteins. In some embodiments, the phage coat protein is protein gIII.In some embodiments, the expression of the nucleic acid sequence of each phage produces an antibody fused to a phage coat protein. In some embodiments, the VH domain further comprises a framework region selected from IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15, and IGHV3-23. In some embodiments, the VL domain further comprises a framework region selected from IGKV1-39, IGKV2-28, IGKV3-15, and IGKV4-1. In some embodiments, the plurality of antibodies comprises at least 7.6×10. 10 In some embodiments, at least 95% of the plurality of antibodies are functional.

[0005] The present invention also provides a method for preparing an antibody library, comprising: a) obtaining sequence information of a plurality of VH-CDR3 and VL-CDR3 sequences from a naive B cell pool, and obtaining sequence information of a plurality of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences from a memory B cell pool; b) assembling a plurality of variable light (VL) domain sequences, each VL domain sequence comprising: a VL-CDR1 sequence obtained from the sequence information from the memory B cells determined in step a; The method comprises the steps of: step 1) assembling a plurality of first nucleic acid sequences encoding a plurality of first antibodies, each of the first antibodies comprising a variable light (VL) domain sequence assembled in step b. and a single fixed heavy chain sequence; d) inserting the plurality of first nucleic acid sequences into a plurality of phages; e) expressing the plurality of first antibodies on the surface of the plurality of phages; f) expressing the plurality of first antibodies on the surface of the plurality of phages; and gating the plurality of first antibodies on the surface of the plurality of phages. g) assembling a plurality of variable heavy (VH) domain sequences, each VH domain sequence comprising: a VH-CDR1 sequence obtained from the sequence information from the memory B cell determined in step a, a VH-CDR2 sequence obtained from the sequence information from the memory B cell determined in step a, and a VH-CDR3 sequence obtained from the sequence information from the memory B cell or the initial B cell determined in step a, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the sequence information from the initial B cell; h) replacing a single fixed heavy chain sequence from the subset of the first nucleic acid sequence with the plurality of VH domain sequences assembled in step g to generate a plurality of second nucleic acid sequences, each second nucleic acid sequence comprising a variable light (VL) domain sequence assembled in step b and a variable heavy (VH) domain sequence assembled in step g, wherein the plurality of second nucleic acid sequences encode a plurality of second antibodies; and i) transforming a plurality of microorganisms with the plurality of phages to produce a plurality of transformants. In some embodiments, the initial B cell pool comprises less than 5% of cells that are not derived from initial B cells. In some embodiments, the memory B cell pool comprises less than 5% of cells that are not derived from memory B cells. In some embodiments, at least one of the VH-CDR3 sequences and VL-CDR3 sequences derived from initial B cells is a naturally occurring sequence. In some embodiments, the VH-CDR3 sequence or VL-CDR3 sequence derived from memory cells is a naturally occurring sequence. In some embodiments, the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence, and VL-CDR2 sequence derived from memory B cells are naturally occurring sequences.In some embodiments, at least one of the VH-CDR3 sequences and VL-CDR3 sequences derived from the initial B cells contains at least 80% sequence homology with the naturally occurring sequence. In some embodiments, the VH-CDR3 sequence or VL-CDR3 sequence derived from the memory cells contains at least 80% sequence homology with the naturally occurring sequence. In some embodiments, the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from the memory B cells contain at least 80% sequence homology with the naturally occurring sequence. In some embodiments, the initial B cell pool, the memory cell pool or a combination thereof are obtained from a plurality of individuals. In some embodiments, a plurality of individuals are at least 50 individuals. In some embodiments, the method further includes sorting the initial B cells and the memory B cells in the sample to produce the initial B cell pool and the memory B cell pool before obtaining the sequence information. In some embodiments, sorting the initial B cells and the memory B cells includes using flow cytometry. In some embodiments, flow cytometry is fluorescence activated cell sorting (FACS). In some embodiments, the method further comprises extracting nucleic acid from the initial B cells and the memory B cells. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the method further comprises reverse transcribing the mRNA into complementary DNA (cDNA). In some embodiments, assembling each VL domain sequence comprises using overlap extension PCR (OE-PCR). In some embodiments, assembling each VH domain sequence comprises using overlap extension PCR (OE-PCR). In some embodiments, the single fixed heavy chain sequence is a germline sequence selected from IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15 and IGHV3-23. In some embodiments, applying at least one selection pressure comprises applying heat stress, selecting with protein A, selecting with protein L, or a combination thereof. In some embodiments, the heat stress is a temperature of at least 65°C. In some embodiments, applying heat stress to multiple phages excludes unstable and easily aggregated phages from a phage subset.

[0006] Also provided herein is an antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the CDR sequence is selected from: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence, wherein the CDR sequence is the same for each antibody in the plurality of antibodies; and (d) a unique combination of the remaining CDR sequences is selected from: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence. In some embodiments, the CDR sequence of (c) is a VH-CDR3 sequence. In some embodiments, the remaining CDR sequences of (d) are VH-CDR1 sequences, VH-CDR2 sequences, VL-CDR1 sequences, VL-CDR2 sequences, and VL-CDR3 sequences. In some embodiments, the CDR sequences of (c) are identical to the CDR sequences derived from the initial antibody clone. In some embodiments, each of the remaining CDR sequences of (d) is present in the antibody library with high diversity. In some embodiments, the high diversity includes at least 1×10 3 In some embodiments, at least one of the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence contains at least 80% sequence homology with a naturally occurring sequence. In some embodiments, the naturally occurring CDR sequence is derived from a human population. In some embodiments, the remaining CDR sequences of (d) exist in a non-naturally occurring combination for each antibody in the plurality of antibodies. In some embodiments, at least one of the plurality of antibodies has at least one of the following: a higher melting temperature (Tm) compared to the initial antibody clone, a higher affinity for the target epitope compared to the initial antibody clone, or a higher cross-reactivity to the target epitope between two or more species compared to the initial antibody clone. In some embodiments, at least one of the plurality of antibodies has a melting temperature (Tm) of about 50°C to about 90°C. In some embodiments, at least one of the plurality of antibodies has a K of 100 nM or less. d Binds to target epitope.

[0007] Further provided herein is a method for generating an antibody library, the method comprising: (a) selecting a CDR sequence, wherein the CDR sequence is selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; (b) replacing the CDR sequence of each antibody in the first antibody library with the CDR sequence selected in (a), thereby generating a second antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (i) the CDR sequence selected in (a); and (ii) a unique combination of the remaining CDR sequences not selected in (a), wherein the remaining CDR sequences are selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence. In some embodiments, the first antibody library comprises a plurality of antibodies, wherein each of the plurality of antibodies comprises a unique combination of VH-CDR1 sequences, VH-CDR2 sequences, VH-CDR3 sequences, VL-CDR1 sequences, VL-CDR2 sequences, and VL-CDR3 sequences. In some embodiments, the CDR sequences selected in (a) are VH-CDR3 sequences. In some embodiments, the remaining CDR sequences of (ii) are VH-CDR1 sequences, VH-CDR2 sequences, VL-CDR1 sequences, VL-CDR2 sequences, and VL-CDR3 sequences. In some embodiments, each of the remaining CDR sequences of (ii) is present in the antibody library with high diversity. In some embodiments, high diversity includes at least 1×10 3 In some embodiments, at least one of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence in the antibody library contains at least 80% sequence homology with a naturally occurring CDR sequence. In some embodiments, the naturally occurring CDR sequence is derived from a human population. In some embodiments, the remaining CDR sequences of (ii) exist in a non-naturally occurring combination for each antibody in a plurality of antibodies. In some embodiments, the CDR sequence of (a) is derived from an initial antibody clone. In some embodiments, at least one antibody in the antibody library has at least one of the following: a higher melting temperature (Tm) compared to the initial antibody clone, a higher affinity for the target epitope compared to the initial antibody clone, or a higher cross-reactivity for the target epitope between two or more species compared to the initial antibody clone. In some embodiments, at least one antibody in the antibody library has a melting temperature (Tm) of about 50°C to about 90°C. In some embodiments, at least one antibody in the antibody library has a K of 100 nM or less. dIn some embodiments, the first antibody library is an antibody library according to any of the aforementioned antibody libraries. In some embodiments, the second antibody library is an antibody library according to any of the aforementioned. In some embodiments, the method further comprises (c) screening the second antibody library for antibodies with desired properties. Incorporation by reference

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided upon request and payment of the necessary fee by the Office. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized, in which:

[0010] Figure 1 Shown are the amounts of VH-CDR3 and VL-CDR3 diversity obtained from naive B cells of a single individual compared to the amounts of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 diversity obtained from memory B cells.

[0011] Figure 2 Shown is the length of time to develop antibody libraries using different technologies including SuperHuman+Carterra and SuperHuman Zero-Day.

[0012] Figure 3 The percentage of clones showing affinity (nM) for PD1 is shown.

[0013] Figure 4 Reactivity of five anti-PD1 clones against human and cynomolgus monkey cell surface PD1 is shown. In the controls, PPE control / parental cells and PPE control / transfected cells are the leftmost peaks in each graph, while the positive control antibody / transfected cells are the rightmost peak. In the selected clone graphs, PPE control / transfected cells are the leftmost peak in each graph, while PPE positive / transfected cells are the rightmost peak.

[0014] Figure 5 Cross-reactivity of the two anti-PD1 clones between humans, mice and cynomolgus monkeys is shown.

[0015] Figure 6 Screening for ligand blocking is shown.

[0016] Figure 7 Shown are beta-galactosidase (bGal) ELISA and Sanger screens of 2 plates containing 61 positive and 49 unique clones.

[0017] Figure 8 The diversity of antibody clone sequences is shown.

[0018] Fig. 9 Antibody fusion variants are shown.

[0019] Fig.10 Variants in VH-CDR1 (CDR-H1) and VH-CDR2 (CDR-H2) of anti-bGal#27 are shown.

[0020] Fig.11 The factors involved in the framework selection strategy are depicted.

[0021] Figure 12A-12B Framework usage of mAbs from Phase I clinical trials is shown. Fig. 12A Heavy chain frameworks used in over 400 mAbs from Phase I clinical trials are shown. Fig. 12B Light chain frameworks used in over 400 mAbs from Phase I clinical trials are shown, showing that the majority of Phase I mAbs are kappa derived.

[0022] Fig.13 Allele frequencies of 12 frameworks in 14 human populations are depicted.

[0023] Fig.14 Allele frequencies of 27 frameworks in 14 human subpopulations are depicted.

[0024] Fig.15 Affinity maturation landscape of human antibody frameworks is shown.

[0025] Fig.16 The amino acid sequences of three framework regions: VH-FR1 (FW1), VH-FR2 (FW2) and VH-FR3 (FW3), and two CDRs: VH-CDR1 (CDR-H1) and VH-CDR2 (CDR-H2) are shown.

[0026] Fig.17 Combinatorial design and selection to generate an antibody library of functionally diverse VH and VK sequences is shown.

[0027] Fig.18 Heavy chain redundancy during various library preparations is shown.

[0028] Fig.19 Sequence overlap between clones is shown.

[0029] Fig. 20 Somatic hypermutations (SHMs) from more than 100 individuals are depicted.

[0030] Fig.21 The frameworks (also referred to herein as scaffolds) used and the diversity of the antibody libraries are described.

[0031] Fig. 22 The characteristics of the antibody library are described.

[0032] Fig.23 Shown are the observed and expected paired mutation frequencies in the VH-CDR1 (CDR-H1) and VH-CDR2 (CDR-H2) regions.

[0033] Fig.24 The positional bias of IGHV3-23 is shown.

[0034] Fig.25 The frequencies of twins are shown.

[0035] Fig.26 Delineated IGHV1-3 allele frequency variation in 14 human populations.

[0036] Fig. 27 It was shown that less than 10,000 clones dominated the total number of clones in peripheral samples from human blood.

[0037] Fig.28 Shown is a phagemid vector expressing an antibody described herein fused to a gIII coat protein.

[0038] Fig.29 Depicted are non-limiting examples of methods for generating the antibody libraries described herein.

[0039] Fig.30 Non-limiting examples of antibody libraries described herein are depicted.

[0040] Fig.31A and Fig.31B Depicted are non-limiting examples of methods for screening the antibody libraries of the disclosure for antibodies with improvements in various properties.

[0041] Fig.32 Depicted are non-limiting exemplary workflows for methods of generating an antibody library as described herein and selecting one or more desired antibodies therefrom.

[0042] Fig.33 Depicted are non-limiting examples of methods for generating the antibody libraries described herein.

[0043] Fig.34 Depicted are non-limiting examples of methods for screening the antibody libraries of the disclosure for antibodies with improvements in various properties.

[0044] Fig.35 Depicted are non-limiting examples of methods for screening the antibody libraries of the disclosure for antibodies with improvements in various properties. DETAILED DESCRIPTION

[0045] A desirable property of an antibody library can be a high degree of functional diversity. Functional diversity can ensure not only that a large number of antibodies are available for testing purposes, but also that this diversity is relevant to function, thereby increasing the utility of these libraries for therapeutic, diagnostic, and research uses. Increased library diversity can be achieved by using naturally occurring complementarity determining regions (CDRs) in non-natural combinations, such as mixing CDRs from memory cells and naive cells, which increases the number of possible CDR combinations. This increase in the functionality of this diversity can be further achieved by selecting for functionality (e.g., the ability to bind to a protein) during antibody library preparation.

[0046] Disclosed herein, in certain cases, are antibodies with unique properties, antibody libraries comprising a high degree of functional diversity, and methods of making the antibodies and antibody libraries. Antibody

[0047] Antibodies can be synthesized by B cells in vivo. Antibody isotypes synthesized by B cells include but are not limited to IgA, IgD, IgE, IgG and IgM. B cells that have not yet encountered an antigen can be referred to as naive B cells, and B cells that have encountered an antigen and are activated by the antigen can be referred to as memory B cells. Naive B cells can express IgM, IgD or a combination thereof. Memory B cells can express IgE, IgA, IgG, IgM or a combination thereof. IgA can be IgA1 or IgA2. IgG can be IgG1, IgG2, IgG3 or IgG4. Memory B cells can be class switching memory B cells or non-converted or marginal zone memory B cells. Non-converted or marginal zone memory B cells can express IgM.

[0048] The complementarity determining region ("CDR") is a portion of the variable region of an immunoglobulin (antibody) that may be responsible for the antigen binding specificity of the antibody. The heavy chain (HC) variable region may include three CDR regions, abbreviated as VH-CDR1, VH-CDR2, and VH-CDR3, and are present in this order on the heavy chain from the N-terminus to the C-terminus; and the light chain (LC) variable region may include three CDR regions, abbreviated as VL-CDR1, VL-CDR2, and VL-CDR3, and are present in this order on the light chain from the N-terminus to the C-terminus. In addition, the light chain may be a kappa chain (VK) or a lambda chain (Vλ). Surrounding and interspersed between the CDRs are framework regions, which may contribute to the structure and may show less variability than the CDR regions.

[0049] The heavy chain variable region may include four framework regions, abbreviated as VH-FR1, VH-FR2, VH-FR3 and VH-FR4. The heavy chain may include from N-terminus to C-terminus: VH-FR1::VH-CDR1::VH-FR2::VH-CDR2::VH-FR3::VH-CDR3::VH-FR4. The light chain variable region may include four framework regions, abbreviated as VL-FR1, VL-FR2, VL-FR3 and VL-FR4. The light chain may include from N-terminus to C-terminus: VL-FR1::VL-CDR1::VL-FR2::VL-CDR2::VL-FR3::VL-CDR3::VL-FR4. In some cases, a "CDR sequence" as used herein refers to a CDR sequence selected from the group consisting of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3, and any combination thereof. Radically diverse antibody library

[0050] The antibody library described herein may comprise a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (a) at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from a naive B cell; (b) if only one of the VH-CDR3 and the VL-CDR3 is derived from a naive B cell, the VH-CDR3 or VL-CDR3 that is not derived from a naive B cell is derived from a memory cell; and (c) the VH-CDR1 sequence, the VH-CDR2 sequence, the VL-CDR1 sequence, and the VL-CDR2 sequence are derived from a memory cell. The antibody library may also be referred to herein as a SuperHuman library.

[0051] In some cases, the multiple antibodies in the antibody library have a high degree of functional diversity.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 80%, 85%, 90%, 95% or 99% of the multiple antibodies are functional.A functional antibody may be an antibody with the ability to bind to a protein.The ability of an antibody to bind to a protein may be determined by screening the antibody for protein A or protein L.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 80% of the multiple antibodies are functional.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 85% of the multiple antibodies are functional.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 90% of the multiple antibodies are functional.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 95% of the multiple antibodies are functional.An antibody library with a high degree of functional diversity may include multiple antibodies, wherein at least 99% of the multiple antibodies are functional.

[0052] The antibody library may contain at least 1.0×10 5 , 2.0×10 5 , 3.0×10 5 4.0×10 5 , 5.0×10 5 , 6.0×10 5 , 7.0×10 5 8.0×10 5 9.0×10 5 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 8.0×10 10 or 9.0×10 10 The antibody library may contain at least 1.0×10 5 The antibody library may contain at least 7.0×10 10 The antibody library can contain at least 7.1×10 10 , 7.2×10 10 , 7.3×10 10 , 7.4×10 10 , 7.5×10 10 , 7.6×10 10 , 7.7×10 10 , 7.8×10 10 or 7.9×1010 The antibody library can contain at least 7.6×10 10 Antibodies.

[0053] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 80% of the plurality of antibodies are functional.

[0054] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 85% of the plurality of antibodies are functional.

[0055] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 90% of the plurality of antibodies are functional.

[0056] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 95% of the plurality of antibodies are functional.

[0057] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×1010 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 99% of the plurality of antibodies are functional.

[0058] The antibodies of the library may comprise a non-natural combination of naturally occurring CDRs, such as a combination of CDRs that are derived from naturally occurring memory B cells and naive B cells, but that do not occur naturally in combination on the same antibody. For example, a non-natural combination of naturally occurring CDRs may comprise at least one CDR derived from naive cells, and the remaining CDRs may be derived from memory cells. For example, a non-natural combination of naturally occurring CDRs may comprise at least one CDR derived from a cell that is primarily a naive B cell source, and the remaining CDRs may be derived from cells that are primarily a memory B cell source. A naturally occurring CDR may refer to a CDR that occurs naturally in a human population.

[0059] The non-naturally occurring combination of naturally occurring CDRs may include at least one CDR derived from a naive cell, while the remaining CDRs are derived from a memory cell. In some cases, at least VL-CDR1 is derived from a naive cell. In some cases, at least VL-CDR2 is derived from a naive cell. In some cases, at least VL-CDR3 is derived from a naive cell. In some cases, at least VH-CDR1 is derived from a naive cell. In some cases, at least VH-CDR2 is derived from a naive cell. In some cases, at least VH-CDR3 is derived from a naive cell.

[0060] The non-natural combination of naturally occurring CDR can include two, three, four or five CDRs derived from initial cells, and the remaining CDRs can be derived from memory cells. For example, two CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, three CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, four CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, five CDRs from the following group of CDRs can be derived from naive cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3, while the remaining CDRs can be derived from memory cells.

[0061] In another non-limiting example of a non-naturally occurring combination, VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR2 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 and VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, and VL-CDR2 may be derived from a memory cell.

[0062] The amino acid residues in the antibody sequence, the variable heavy chain sequence of the antibody or the variable light chain sequence of the antibody can be referred to according to their Kabat positions. As used herein, "Kabat position" can refer to the numbering system described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH, USA. In some cases, the antibodies described herein include variations at Kabat position H93, Kabat position H94, or a combination thereof. In some cases, at least one antibody in the antibody library includes variations at Kabat position H93, Kabat position H94, or a combination thereof. In some cases, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the antibodies in the antibody library include variations at Kabat position H93, Kabat position H94, or a combination thereof. Variations can be mutations, insertions or deletions.

[0063] When used with respect to a sequence, "derived from" can refer to any CDR sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence homology to a naturally occurring CDR sequence. "Derived from" can refer to any CDR sequence obtained from sequencing information obtained from a pool of cells that are primarily naive B cell-derived or a pool of cells that are primarily memory B cell-derived. For example, a sequence is "derived from" a cell if (1) a sequence is observed in the cell and (2) the same sequence (or a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence homology to the sequence) is chemically synthesized based on the observed sequence.

[0064] The VH-CDR1 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR1 sequence derived from a naive B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell.

[0065] The VL-CDR1 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR1 sequence derived from the initial B cell may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may be a synthetic VL-CDR2 sequence. The VL-CDR2 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell. The VL-CDR3 sequence derived from a naive B cell may be a synthetic VL-CDR3 sequence. The VL-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell.

[0066] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The naive B cell pool can be obtained from multiple individuals. The naive B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from naive B cells.

[0067] The VH-CDR1 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR1 sequence derived from a memory B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell.

[0068] The VL-CDR1 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR1 sequence derived from memory B cells may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell. The VL-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell.

[0069] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The memory B cell pool can be obtained from multiple individuals. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from memory B cells. The memory B cells may be CD27+ B cells. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from CD27+ B cells.

[0070] The percentage of sequence homology can be calculated by determining the number of positions at which the same nucleic acid base occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions, which may include additions or deletions, and then multiplying the result by 100 to obtain the percentage of sequence homology. The percentage of sequence homology, also known as the percentage of sequence identity, can be determined by aligning each sequence in any suitable sequence alignment program, such as Clustal Omega, Multiple Sequence Comparison by Logarithmic Expectations (MUSCLE), Multiple Alignment Using Fast Fourier Transforms (MAFFT), MegAlign, and Basic Local Alignment Search Tool (BLAST).

[0071] The naive cell may be a naive B cell. The naive B cell may be a human naive B cell. The memory cell may be a memory B cell. The memory B cell may be a human memory B cell. In some cases, the naive B cell exhibits increased diversity of VH-CDR3 and VL-CDR3 sequences compared to the VH-CDR3 and VL-CDR3 sequences from the memory B cell ( Figure 1 ). The naive cells and memory cells can be obtained from a biological sample, such as blood, from an individual or multiple individuals. The naive cells and memory cells can be physically separated from the sample using markers specific to the naive cells or memory cells.

[0072] Markers can be used to identify, separate or sort B cells, naive B cells and memory B cells from biological samples. Examples of markers for identifying, separating or sorting B cells include, but are not limited to, CD19+. Examples of markers for identifying, separating or sorting naive B cells include, but are not limited to, CD19+, CD27-, IgD+, IgM+ and combinations thereof. Examples of markers for identifying, separating or sorting memory B cells include, but are not limited to, CD19+, CD27+ and combinations thereof. In some embodiments, memory B cells are sorted using CD27+. Examples of markers for identifying, separating or sorting class-converted memory B cells include, but are not limited to, CD19+, CD27+, CD27+, IgD-, IgM- and combinations thereof. Examples of markers for identifying, separating or sorting non-converted or marginal zone memory B cells include, but are not limited to, CD19+, CD27+, IgD+, IgM+ and combinations thereof. In some cases, memory B cells can be identified, separated or sorted with the following markers: CD19+, CD27+, IgD-, IgM+ and combinations thereof. The naive cell from which VH-CDR3 is derived may be a CD27- / IgM+ B cell. The memory cell from which VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2 and VL-CDR3 are derived may be a CD27+ / IgG+ B cell.

[0073] The CDR sequences of antibodies can be CDR sequences found in naive B cells and memory B cells found in a single or multiple individuals. The individual can be a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a pig, a goat, a rabbit, a horse, a cow, a cat or a dog. In some cases, the CDR sequences are CDR sequences obtained from publicly available sources. Examples of sources of publicly available CDR sequences include SAbDab (http: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / Welcome.php) and PylgClassify (http: / / dunbrack2.fccc.edu / PyIgClassify / ).

[0074] Germline antibody sequences may include germline framework and germline CDR sequences. Each CDR in the antibodies found in the antibody library may include at least 1, 2, 3 or 4 mutations compared to the corresponding germline CDR regions. Each CDR in the antibodies in the antibody library may include no more than 4 mutations compared to the corresponding framework CDR regions.

[0075] The framework of the antibody may be a naturally occurring framework. A naturally occurring framework may be a framework found in a mammal. The mammal may be a primate, mouse, rat, pig, goat, rabbit, horse, cow, cat or dog. The primate may be a human. The framework may contain at least one variant compared to a naturally occurring framework. A variant may be a mutation, insertion or deletion. A variant may be a variant found in a nucleic acid sequence encoding the antibody or a variant found in an amino acid sequence of the antibody. Any suitable framework sequence may be used, such as those previously used in Phase I clinical trials ( Fig. 12A , Fig. 12B As used herein, the framework of an antibody may refer to the framework regions of the variable heavy chain (VH-FR1, VH-FR2, VH-FR3, and VH-FR4), the framework regions of the variable light chain (VL-FR1, VL-FR2, VL-FR3, and VL-FR4), or a combination thereof. The framework regions of the antibodies in the antibody library may be the same as the germline framework regions.

[0076] The framework can be the best framework for treatment. The best framework for treatment can include at least one, at least two, at least three, at least four, at least five, at least six or all of the following properties selected from the group: a) safety previously demonstrated in human monoclonal antibodies, b) thermal stability; c) not prone to aggregation; d) including a single dominant allele at the amino acid level in the entire human population; e) including different typical topologies of CDR; f) well expressed in bacteria; and g) well displayed on phage. The framework with safety previously demonstrated in human monoclonal antibodies can be the framework of antibodies that have been used in at least Phase I clinical trials. A thermally stable framework can be a framework that is stable at at least 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or above 100°C. A thermally stable framework can be a framework that can withstand a temperature increase of at least 3°C ​​per minute, 4°C per minute or 5°C per minute. A framework that expresses well in bacteria can be a framework that produces biologically active antibodies in bacteria. The bacterium may be Escherichia coli (E. coli). The bacterium may be an engineered bacterium. The bacterium may be a bacterium optimized for antibody expression. A framework that displays well on a phage may be a framework that produces a biologically active antibody when displayed on the surface of a phage.

[0077] Examples of strategies for selecting frames are described in Fig.11 In which the ideal framework of the antibody can be an antibody that shows structural diversity, has been successfully used in Phase I clinical trials in humans, has low immunogenicity, shows aggregation resistance, shows adaptability, and is thermally stable. In some cases, if the antibody framework has an inherent autoreactivity to blood cells (e.g., IGHV4-34), has poor stability characteristics (e.g., IGHV2-5), has a V gene that is not found in at least 50% of individuals (e.g., IGHV4-b), shows a V gene that tends to aggregate (e.g., IGLV6-57), or a combination thereof, the antibody framework is avoided.

[0078] The amino acid sequences of the antibody frameworks herein may contain more than one dominant allele, wherein different dominant alleles exist in different human populations ( Fig.13 and Fig.14 For example, the IGHV1-3 framework contains three alleles: IGVH1-3*01, IGVH1-3*02, and IGVH1-3*03, which are found at different frequencies in different human populations ( Fig.26). In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in at least two human populations. In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in all human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least two human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least twelve human populations. In some cases, the framework region of the VH domain is a framework region from IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15, or IGHV3-23. In some cases, the framework region of the VH domain is a framework region from IGHV2-5, IGHV3-7, IGVH4-34, IGHV5-51, IGHV1-24, IGHV2-26, IGHV3-72, IGHV3-74, IGHV3-9, IGHV3-30, IGHV3-33, IGHV3-53, IGHV3-66, IGHV4-30-4, IGHV4-31, IGHV4-59, IGHV4-61, or IGHV5-51. In some cases, the framework region of the VH domain of the antibodies in the antibody library is a framework region from IGHV1-46, IGHV3-23, or a combination thereof. In some cases, the framework regions of the VL domains of the antibodies in the antibody library are framework regions from IGKV1-39, IGKV2-28, IGKV3-15, IGKV4-1, IGKV1-5, IGKV1-12, IGKV1-13, IGKV3-11, IGKV3-20, or a combination thereof. In one example, a subset of antibodies in the antibody library may have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV1-39, while the remaining antibodies in the antibody library have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV2-28.

[0079] In some cases, disclosed herein are nucleic acid sequences encoding antibodies described herein. The nucleic acid sequence may be a DNA or RNA sequence. The nucleic acid may be inserted into a vector. The vector may be a bacteriophage. The bacteriophage may be a phagemid or a bacteriophage. The phagemid may be pMID21. The bacteriophage may be DY3F63, M13 phage, fd filamentous phage, T4 phage, T7 phage, or lambda phage. In some cases, the phagemid may be combined with a bacteriophage (i.e., a "helper" phage) and introduced into a microorganism. The microorganism may be a filamentous bacterium. The filamentous bacterium may be Escherichia coli.

[0080] The antibody library described herein comprises a plurality of antibodies. The plurality of antibodies may be at least 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 , 8.0×10 10 9.0×10 10 or 10.0×10 10 The plurality of antibodies may be at least 1.0×10 11 The plurality of antibodies may be at least 7.6×10 10 Due to the high diversity of such libraries, they can be unique. For example, in any library herein, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the plurality of antibodies can be unique. In some cases, the library has more than 7.0×10 10 The invention relates to a method for preparing a plurality of antibodies, wherein at least 20% of the plurality of antibodies are unique. Relative to other antibodies in the antibody library, a unique antibody can differ by at least one nucleic acid or at least one amino acid residue.

[0081] The total amount of antibodies found naturally in the human body (e.g., from naive and memory cells) as well as antibody libraries generated by other library preparation methods can contain highly redundant heavy chain sequences ( Fig.18 ). If one heavy chain sequence of an antibody in the library is redundant with another heavy chain sequence of a different antibody, it can be indicated that the heavy chain sequences are identical. Two or more antibodies with redundant heavy chain sequences can contain different antibody framework regions, different light chain sequences, or a combination thereof. The antibody library produced by the methods described herein can show reduced heavy chain redundancy. The reduction in heavy chain redundancy can increase the diversity of the antibody library. Redundancy can be measured by the percentage of the library occupied by the top clones. The antibody library produced herein can have a redundancy of about 2%, about 3%, about 4%, or about 5%. Fig.18 In some cases, the maximum number of heavy chains in traditional natural libraries is limited to 1.0 × 10 due to the combination of naturally occurring CDRs. 7In some cases, the heavy chains of the antibodies in the libraries described herein are not restricted by the combinations of naturally occurring CDRs and may contain more than 1.0×10 11 Antibodies.

[0082] In some cases, the antibody library is Fig.21 In some cases, the antibody library is as described in Fig. 22 The antibody library described in . Methods for generating diverse antibody libraries

[0083] In some cases, described herein are methods for preparing an antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (a) at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from a naive B cell; (b) the VH-CDR3 sequence or the VL-CDR3 sequence that is not derived from a naive B cell is derived from a memory B cell; and (c) the VH-CDR1 sequence, the VH-CDR2 sequence, the VL-CDR1 sequence, and the VL-CDR2 sequence are derived from a memory cell.

[0084] In some cases, the methods described herein produce antibody libraries with high functional diversity.Antibody libraries with high functional diversity can include multiple antibodies, wherein at least 80%, 85%, 90%, 95% or 99% of the multiple antibodies are functional.A functional antibody can be an antibody with the ability to bind to a protein.The ability of an antibody to bind to a protein can be determined by screening the antibody for protein A or protein L.Antibody libraries with high functional diversity can include multiple antibodies, wherein at least 90% of the multiple antibodies are functional.Antibody libraries with high functional diversity can include multiple antibodies, wherein at least 95% of the multiple antibodies are functional.Antibody libraries with high functional diversity can include multiple antibodies, wherein at least 99% of the multiple antibodies are functional.

[0085] The antibody library may contain at least 1.0×10 5 , 2.0×10 5 , 3.0×10 5 4.0×10 5 , 5.0×10 5 , 6.0×10 5 , 7.0×10 5 8.0×10 5 9.0×10 5 , 1.0×1010 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 , 8.0×10 10 or 9.0×10 10 The antibody library may contain at least 1.0×10 5 The antibody library may contain at least 7.0×10 10 The antibody library can contain at least 7.1×10 10 , 7.2×10 10 , 7.3×10 10 , 7.4×10 10 , 7.5×10 10 , 7.6×10 10 , 7.7×10 10 , 7.8×10 10 or 7.9×10 10 The antibody library can contain at least 7.6×10 10 Antibodies.

[0086] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 80% of the plurality of antibodies are functional.

[0087] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 85% of the plurality of antibodies are functional.

[0088] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 90% of the plurality of antibodies are functional.

[0089] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 95% of the plurality of antibodies are functional.

[0090] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 99% of the plurality of antibodies are functional.

[0091] The method for preparing an antibody library may include: (a) obtaining sequence information of a plurality of VH-CDR3 and VL-CDR3 sequences from a naive B cell pool and obtaining sequence information of a plurality of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences from a memory B cell pool; (b) assembling a plurality of variable light (VL) domain sequences, each VL domain sequence comprising: a VL-CDR1 sequence obtained from the sequence information from the memory B cells determined in step a, a VL-CDR2 sequence obtained from the sequence information from the memory B cells determined in step a, (c) assembling a plurality of first nucleic acid sequences encoding a plurality of first antibodies, each of the first antibodies comprising: (i) a variable light (VL) domain sequence assembled in step b; and (ii) a single fixed heavy chain sequence; (d) inserting the plurality of first nucleic acid sequences into a plurality of phages; (e) expressing the plurality of first antibodies on the surface of the plurality of phages; and (f) The phage exerts at least one selection pressure to produce a subset of phage comprising a subset of the first nucleic acid sequence; (g) assembling a plurality of variable heavy (VH) domain sequences, each VH domain sequence comprising: a VH-CDR1 sequence obtained from the sequence information from the memory B cell determined in step a, a VH-CDR2 sequence obtained from the sequence information from the memory B cell determined in step a, and a VH-CDR3 sequence obtained from the sequence information from the memory B cell or the naive B cell determined in step a, wherein the VH-CDR3 sequence and the VL- At least one of the CDR3 sequences is derived from sequence information from an initial B cell; (h) replacing a single fixed heavy chain sequence from a subset of first nucleic acid sequences with a plurality of VH domain sequences assembled in step g to generate a plurality of second nucleic acid sequences, each second nucleic acid sequence comprising: (i) a variable light (VL) domain sequence assembled in step b, and (ii) a variable heavy (VH) domain sequence assembled in step g, wherein the plurality of second nucleic acid sequences encode a plurality of second antibodies; and (i) transforming a plurality of microorganisms with a plurality of bacteriophages to produce a plurality of transformants.

[0092] The method may include obtaining samples containing naive B cells and memory B cells from multiple individuals. The sample may be blood, plasma, or serum. A peripheral sample of human blood may contain hundreds of thousands of memory clones and plasmablasts, of which a collection of less than 10,000 dominates the sample ( Fig. 27). The plurality of individuals may be a plurality of mammals. The plurality of mammals may be a plurality of primates, mice, rats, pigs, goats, rabbits, horses, cows, cats, or dogs. The plurality of mammals may be a plurality of humans. The plurality of individuals may be at least 25, 50, 75, 100, 125, or 150 individuals. The plurality of individuals may be 50-100 individuals. The plurality of individuals may be 50-140 individuals. The plurality of individuals may be at least 50 individuals. The plurality of individuals may be at least 140 individuals. The sample comprising naive B cells and memory B cells from an individual may comprise at least about 5×10 7 Initial B cell clones and 5×10 5 memory B cell clones.

[0093] The method may include sorting or separating the initial B cells and memory B cells in the sample before obtaining the sequence information. The memory B cells may be CD27+ B cells. Therefore, the sequence information may include separate sequence information for the initial B cells and the memory B cells. Sorting the initial B cells and the memory B cells may include using flow cytometry. In some cases, flow cytometry is fluorescence activated cell sorting (FACS). Sorting the initial B cells and the memory B cells may include an immunomagnetic cell separation procedure based on markers present on the surface of the initial B cells or the memory B cells. Sorting the initial B cells and the memory B cells from the sample may produce an initial B cell pool and a memory B cell pool. Sorting the initial B cells and the memory B cells from the sample may produce a plurality of initial B cell pools and a plurality of memory B cell pools. The initial B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from the initial B cells. A pool of naive B cells comprising less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from naive B cells may also be referred to herein as a pool that is primarily derived from naive B cells. A pool of memory B cells may comprise less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from memory B cells. A pool of memory B cells comprising less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from memory B cells may also be referred to herein as a pool that is primarily derived from memory B cells.

[0094] In some cases, the quality of the memory B cell pool or the initial B cell pool will be checked using next generation sequencing (NGS). Pools with problematic diversity or biochemical liability can be discarded. Examples of biochemical liability include, but are not limited to, N-linked glycosylation, deamination, acid hydrolysis, positively charged internal peptide cleavage, free cysteine, free methionine, alternative stop codons, hidden splice sites, tev cleavage sites, and excessively positively charged CDRs. In some cases, sequence data from at least one individual is removed from the pool. If the sequence data has problematic diversity or biochemical liability, sequence data from an individual can be removed from the pool.

[0095] The method may include extracting nucleic acid from naive B cells and extracting nucleic acid from memory B cells. After separating or isolating the naive cells and memory cells from the sample, nucleic acid may be extracted from each of the naive cells and memory cells. The nucleic acid may be DNA or messenger RNA (mRNA). If the nucleic acid is mRNA, the method may further include reverse transcribing the mRNA into complementary DNA (cDNA).

[0096] The method for preparing an antibody library may include obtaining sequence information of multiple VH-CDR3 and VL-CDR3 sequences from naive B cells from multiple individuals and obtaining sequence information of multiple VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences from memory B cells from multiple individuals. The sequence information from naive B cells can be obtained from a pool of naive B cells. The sequence information from memory B cells can be obtained from a pool of memory B cells. Obtaining sequence information of CDR sequences may include sequencing the CDR sequences. Sequencing of multiple CDR sequences may include any suitable sequencing technology, such as next generation sequencing (NGS) or Sanger sequencing. Examples of next generation sequencing include, but are not limited to, pyrophosphate sequencing, synthetic sequencing, ligation sequencing and single molecule sequencing. Sequencing of multiple CDR sequences may generate sequence information.

[0097] Sequencing a plurality of VH-CDR and VL-CDR sequences may include sequencing nucleic acid extracted from naive B cells from the sample and nucleic acid extracted from memory B cells from the sample, respectively.

[0098] Assembling or synthesizing a VH sequence or a VL sequence may include using overlap extension PCR (OE-PCR). In some cases, overlapping fragments comprising a portion of a CDR of a VH domain or a CDR of a VL domain are generated. Multiple overlapping fragments comprising a portion of a CDR of a VH domain or a CDR of a VL domain may cover all of the CDRs of a VH domain sequence or a CDR of a VL domain sequence. Overlapping fragments may be dsDNA fragments. OE-PCR may include assembling overlapping fragments to generate all of the CDRs of a VH domain or a CDR of a VL domain. The CDR of a VH domain may be VH-CDR1, VH-CDR2, VH-CDR3, or a combination thereof. The CDR of a VL domain may be VL-CDR1, VL-CDR2, VL-CDR3, or a combination thereof. VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, VL-CDR3 or a combination thereof may be synthesized to contain at least one of the following properties: (a) being a CDR sequence derived from a human germline sequence, (b) containing no more than 4 amino acid mutations compared to the germline sequence, (c) being: (i) identified as naturally occurring in at least 2 individuals and enriched without fitness disadvantage or (ii) substantially enriched during panning; (d) not containing any biochemical bias; or (e) a combination thereof. The germline sequence may be IGHJ4, IGHV1-69, IGHV1-46, IGHV3-23, IGKV1-39, IGKV2-28, IGKV3-15 or IGKV4-1, or a combination thereof.

[0099] The VH sequence, VL sequence or a combination thereof assembled using OE-PCR can be cloned into a vector. The vector can be a phage. The phage can be a bacteriophage or a phagemid. The vector can be a HuCAL phage. The vector can further comprise a gene encoding a surface coat protein. The surface coat protein can be pIII, pVIII, pVI, pVII, pIX or gIII protein. The surface coat protein can be a gIII protein. In some cases, the expression of the antibody encoded by the vector includes the expression of the antibody fused to the surface coat protein of the vector. The expressed antibody can be displayed on the surface of the vector.

[0100] The method for preparing an antibody library can include assembling multiple VL domain sequences, each of the multiple VL domain sequences comprising: a VL-CDR1 sequence derived from sequence information from a memory B cell, a VL-CDR2 sequence derived from sequence information from a memory B cell, and a VL-CDR3 sequence derived from sequence information from a memory B cell or a naive B cell. The VL domain sequence can be cloned into a vector in combination with a single fixed heavy chain sequence. The single fixed heavy chain sequence can be IGHV3-23 or IGHJ4. The single fixed heavy chain sequence can be referred to as a filler sequence.

[0101] The method for preparing an antibody library may include assembling a plurality of first nucleic acid sequences encoding a plurality of first antibodies, each of the plurality of first antibodies comprising: a variable light (VL) domain sequence; and a single fixed heavy chain sequence. The method for preparing an antibody library may include inserting the plurality of first nucleic acid sequences into a plurality of vectors. The vector may be a phage. The antibodies encoded by the vector comprising the assembled VL domains and the single fixed heavy chain sequence may be expressed in the vector. The antibodies may be expressed on the surface of the vector.

[0102] The method for preparing an antibody library may include applying at least one selection pressure to a plurality of carriers, wherein each of the plurality of carriers expresses an antibody. After applying the selection pressure, a subset of phages that can withstand the selection pressure may be produced. The selection pressure may be applying heat stress, selecting with protein A, selecting with protein L, or a combination thereof. Heat stress may be a temperature of about 65°C. Heat stress may be a temperature of at least 30°C, 40°C, 50°C, 60°C, 70°C, or 80°C. In some cases, if the carrier is unstable or prone to aggregation, heat stress is applied to the carrier to exclude the carrier. In some cases, if the carrier expresses an antibody that does not have the ability to bind to a protein, the selection of protein A or protein L is applied to exclude the carrier. The selection of protein A or protein L may allow the selection of antibodies that bind to the protein to be allowed. The antibody that binds to the protein may be thermostable. In some cases, after selecting the antibody that binds to the protein, the nucleotide sequence corresponding to the antibody that binds to the protein may be determined.

[0103] The method for preparing an antibody library may include assembling a plurality of VH domain sequences, wherein each VH domain sequence includes: a VH-CDR1 sequence derived from sequence information from a memory B cell, a VH-CDR2 sequence derived from sequence information from a memory B cell, and a VH-CDR3 sequence derived from sequence information from a memory B cell or a naive B cell. The VH-CDR1 sequence and the VH-CDR2 sequence may be sequences obtained from memory B cells, while the VH-CDR3 sequence may be a sequence from a naive B cell. If the vector can successfully withstand the applied selection pressure, the assembled VH domain sequence may be used to replace a single fixed heavy chain sequence. For each of the plurality of antibodies, at least one of the VH-CDR3 sequence and the VL-CDR3 sequence may be derived from sequence information from a naive B cell.

[0104] The vector comprising the assembled VL domain and the assembled VH domain can be transformed into a microorganism. The microorganism can be a bacterium. The bacterium can be a filamentous bacterium. The filamentous bacterium can be Escherichia coli. The microorganism can be any suitable commercially available strain.

[0105] The vector can be transformed into the microorganism using electroporation, chemical transformation, heat shock transformation, or a combination thereof.

[0106] Electroporation can include applying a high voltage electric field to a ligated mixture containing a microorganism to be transformed and a vector. The high voltage can range from 1 to 25 kV / cm. The high voltage can range from 3 to 24 kV / cm. Examples of high voltages that can be applied to a microorganism to induce transformation include, but are not limited to, 10 kV / cm, 15 kV / cm, 20 kV / cm, and 25 kV / cm. The high voltage can be applied as a pulse or multiple pulses. Multiple pulses can be high voltage pulses applied every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, or 1000 microseconds (μs). Multiple pulses can be high voltage pulses applied every 10, 20, 30, 30, 50, 60, 70, 80, 90, or 100 milliseconds (msec). Electroporation can be applied to a microorganism at room temperature or 4°C.

[0107] The vector can be purified and resuspended in water or TE before being added to the ligation mixture. The ligation mixture can include a buffer. Examples of buffers include, but are not limited to, phosphate buffered saline (PBS), hepes buffer (HBSS), or culture medium. The buffer can be a hypotonic buffer. The buffer can be a high resistance buffer. In some cases, after electroporation, recovery medium is added to the ligation mixture.

[0108] Chemical transformation may include incubating the microorganism and the carrier with a cation. The cation may be Mg2+, Mn2+, Rb+, or Ca2+. Chemical transformation may include incubating the microorganism and the carrier with CaCl2, MgCl2, MnCl2, or RbCl.

[0109] Heat shock transformation can include applying high temperature to the microorganism and carrier to induce transformation. The high temperature can be 42°C. The temperature can be applied for 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds or 1 minute. Heat shock can be applied before, during or after electroporation or chemical transformation.

[0110] The vector may comprise a selection marker. The selection marker may be an antibiotic resistance gene or an optical selection marker, such as green fluorescent protein. The antibiotic resistance gene may confer resistance to an antibody selected from the group consisting of kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erthyromycin, polymyxin B, tetracycline, chloramphenicol, and combinations thereof to the microorganism transformed by the vector. The selection marker may allow for the exclusion of microorganisms that have not been transformed by the vector.

[0111] The microorganisms transformed with the vector can be referred to as transformants. The generation of multiple antibodies can include the generation of multiple transformants. In some cases, the multiple transformants include at least 7.6×10 10 transformants. At least 7.6×10 10 Each transformant can contain 0.5×10 10 VH-CDR3 sequences. In some cases, at least 20% of the plurality of transformants are unique. For example, if the plurality of transformants includes 7.6×10 10 transformants, then in some embodiments, at least 1.52×10 10 A transformant is unique. A unique transformant can be a transformant with a unique or non-redundant sequence compared to other transformants in a plurality of transformants. In some cases, the antibody library described herein can be screened to obtain antibodies specific to any desired target. Examples of targets include, but are not limited to, PD1, LAG3, OX40, CTLA4, SIRPA, CD47, VISTA, 41BB, TIM3, GITR, ICOS, TIGIT, GHR, HGH, amyloid beta, alpha synuclein, Tau, and beta secretase. The length of time for developing the antibody library described herein can be less than 2 months, less than 1 month, or less than 2 weeks. In one example, the development of the antibody library can be less than 2 months, including the time involved in panning, screening, and optimization ( Figure 2 In another example, the length of time may be less than 2 weeks ( Figure 2 ). Antibody optimization and resulting library (Tumbler library)

[0112] In some aspects, the antibody library described herein may comprise a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the CDR sequence is selected from the following: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence, wherein the CDR sequence of each antibody in the plurality of antibodies is the same; and (d) a unique combination of the remaining CDR sequences is selected from the following: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3. Antibody libraries may also be referred to herein as Tumbler libraries.

[0113] In one example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR1 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the multiple antibodies. In some cases, the VH-CDR1 sequence is derived from an initial antibody clone.

[0114] In another example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR2 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR1 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the multiple antibodies. In some cases, the VH-CDR2 sequence is derived from an initial antibody clone.

[0115] In another example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR3 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 are present in different combinations in each antibody in the multiple antibodies. In some cases, the VH-CDR3 sequence is derived from an initial antibody clone.

[0116] In another example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR1 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the multiple antibodies. In some cases, the VL-CDR1 sequence is derived from an initial antibody clone.

[0117] In another example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR2 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the multiple antibodies. In some cases, the VL-CDR2 sequence is derived from an initial antibody clone.

[0118] In another example, the antibody library described herein can include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR3 sequence is the same for each antibody in the multiple antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, and the VL-CDR2 sequence are present in different combinations in each antibody in the multiple antibodies. In some cases, the VL-CDR3 sequence is derived from an initial antibody clone.

[0119] In various aspects, each antibody in the antibody library comprises a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence or a VL-CDR3 sequence selected from an initial antibody clone. As used herein, the term "initial antibody clone" may refer to any antibody or antibody fragment with a desired property, such as an affinity for an amino acid sequence of a desired epitope, an amino acid sequence of the antibody or antibody fragment, a nucleotide sequence encoding the antibody or antibody fragment, or any computer-simulated amino acid or nucleotide sequence corresponding to the antibody or antibody fragment. In various aspects, each antibody in the antibody library may comprise the same CDR sequence derived from the initial antibody clone. In addition, each antibody in the antibody library may comprise a different combination of the remaining CDR sequences that are not derived from the initial antibody clone. In some cases, the remaining CDR sequences may be derived from a highly diverse antibody library (e.g., a SuperHuman antibody library as described herein). In some cases, one of the CDRs may be derived from an initial antibody clone, and the remaining CDRs may be derived from a highly diverse antibody library (e.g., a SuperHuman antibody library). In some cases, a highly diverse antibody library may have high diversity in each CDR sequence that is not derived from an initial antibody clone. In a non-limiting example, Fig.29 As depicted, each antibody in the antibody library may comprise a clone derived from an initial antibody clone ( Fig.29 In addition, each antibody in the antibody library may contain a different combination of the remaining CDR sequences (in this example, VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3) that are not derived from the initial antibody clone.

[0120] In various aspects, one or more of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence are naturally occurring. In various aspects, each of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence is naturally occurring, but is present in each antibody in a non-naturally occurring combination. In various aspects, one or more of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence are naturally occurring in the human population or are derived from human CDR sequences. In various aspects, each of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, and VL-CDR3 sequence naturally occurs in the human population, or is derived from a human CDR sequence.

[0121] In various aspects, the antibodies of the library may comprise non-naturally occurring combinations of naturally occurring CDRs, such as combinations of CDRs derived from naturally occurring memory B cells and naive B cells, but their co-occurrence on the same antibody is not naturally occurring. For example, the non-naturally occurring combination of naturally occurring CDRs may comprise at least one CDR derived from a naive cell, while the remaining CDRs may be derived from a memory cell. For example, the non-naturally occurring combination of naturally occurring CDRs may comprise at least one CDR derived from a cell that is primarily a naive B cell source, while the remaining CDRs may be derived from a cell that is primarily a memory B cell source.

[0122] The non-naturally occurring combination of naturally occurring CDRs may include at least one CDR derived from a naive cell, while the remaining CDRs are derived from a memory cell. In some cases, at least VL-CDR1 is derived from a naive cell. In some cases, at least VL-CDR2 is derived from a naive cell. In some cases, at least VL-CDR3 is derived from a naive cell. In some cases, at least VH-CDR1 is derived from a naive cell. In some cases, at least VH-CDR2 is derived from a naive cell. In some cases, at least VH-CDR3 is derived from a naive cell.

[0123] The non-natural combination of naturally occurring CDR can include two, three, four or five CDRs derived from initial cells, and the remaining CDRs can be derived from memory cells. For example, two CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, three CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, four CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, five CDRs from the following group of CDRs can be derived from naive cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3, while the remaining CDRs can be derived from memory cells.

[0124] In another non-limiting example of a non-naturally occurring combination, VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR2 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 and VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, and VL-CDR2 may be derived from a memory cell.

[0125] The VH-CDR1 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR1 sequence derived from a naive B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell.

[0126] The VL-CDR1 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR1 sequence derived from the initial B cell may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may be a synthetic VL-CDR2 sequence. The VL-CDR2 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell. The VL-CDR3 sequence derived from a naive B cell may be a synthetic VL-CDR3 sequence. The VL-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell.

[0127] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The naive B cell pool can be obtained from multiple individuals. The naive B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from naive B cells.

[0128] The VH-CDR1 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR1 sequence derived from a memory B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell.

[0129] The VL-CDR1 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR1 sequence derived from memory B cells may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell. The VL-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell.

[0130] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The memory B cell pool can be obtained from multiple individuals. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from memory B cells. The memory B cells may be CD27+ B cells. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from CD27+ B cells.

[0131] The naive cell may be a naive B cell. The naive B cell may be a human naive B cell. The memory cell may be a memory B cell. The memory B cell may be a human memory B cell. In some cases, the naive B cell exhibits increased diversity of VH-CDR3 and VL-CDR3 sequences compared to the VH-CDR3 and VL-CDR3 sequences from the memory B cell ( Figure 1 ). The naive cells and memory cells can be obtained from a biological sample, such as blood, from an individual or multiple individuals. The naive cells and memory cells can be physically separated from the sample using markers specific to the naive cells or memory cells.

[0132] Markers can be used to identify, separate or sort B cells, naive B cells and memory B cells from biological samples. Examples of markers for identifying, separating or sorting B cells include, but are not limited to, CD19+. Examples of markers for identifying, separating or sorting naive B cells include, but are not limited to, CD19+, CD27-, IgD+, IgM+ and combinations thereof. Examples of markers for identifying, separating or sorting memory B cells include, but are not limited to, CD19+, CD27+ and combinations thereof. In some embodiments, CD27+ is used to sort memory B cells. Examples of markers for identifying, separating or sorting class-converted memory B cells include, but are not limited to, CD19+, CD27+, CD27+, IgD-, IgM- and combinations thereof. Examples of markers for identifying, separating or sorting non-converted or marginal zone memory B cells include, but are not limited to, CD19+, CD27+, IgD+, IgM+ and combinations thereof. In some cases, memory B cells can be identified, separated or sorted with the following markers: CD19+, CD27+, IgD-, IgM+ and combinations thereof. The naive cell from which VH-CDR3 is derived may be a CD27- / IgM+ B cell. The memory cell from which VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2 and VL-CDR3 are derived may be a CD27+ / IgG+ B cell.

[0133] The CDR sequences of antibodies can be CDR sequences found in naive B cells and memory B cells found in a single or multiple individuals. The individual can be a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a pig, a goat, a rabbit, a horse, a cow, a cat or a dog. In some cases, the CDR sequences are CDR sequences obtained from publicly available sources. Examples of sources of publicly available CDR sequences include SAbDab (http: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / Welcome.php) and PylgClassify (http: / / dunbrack2.fccc.edu / PyIgClassify / ).

[0134] In various aspects, each antibody in the antibody library can comprise the same scaffold, e.g., the same combination of framework sequences (see Fig.30). In various aspects, the VH domain of each antibody in the antibody library comprises a VH-FR1 sequence, a VH-FR2 sequence, a VH-FR3 sequence, and a VH-FR4 sequence. In some cases, each of the VH-FR1 sequence, the VH-FR2 sequence, the VH-FR3 sequence, and the VH-FR4 sequence of each antibody in the antibody library is identical. In some cases, each of the VH-FR1 sequence, the VH-FR2 sequence, the VH-FR3 sequence, and the VH-FR4 sequence is derived from the initial antibody clone from which the CDR sequence is derived (see Fig.30 ). In some cases, the VH-FR1 sequence can be the same VH-FR1 sequence as the initial antibody clone. In some cases, the VH-FR2 sequence can be the same VH-FR2 sequence as the initial antibody clone. In some cases, the VH-FR3 sequence can be the same VH-FR3 sequence as the initial antibody clone. In some cases, the VH-FR4 sequence can be the same VH-FR4 sequence as the initial antibody clone. In various aspects, the VL domain of each antibody in the antibody library comprises a VL-FR1 sequence, a VL-FR2 sequence, a VL-FR3 sequence, and a VL-FR4 sequence. In some cases, each of the VL-FR1 sequence, the VL-FR2 sequence, the VL-FR3 sequence, and the VL-FR4 sequence of each antibody in the antibody library is the same. In some cases, each of the VL-FR1 sequence, the VL-FR2 sequence, the VL-FR3 sequence, and the VL-FR4 sequence can be derived from the initial antibody clone from which the CDR sequence is derived (see Fig.30 ). In some cases, the VL-FR1 sequence can be the same VL-FR1 sequence as the initial antibody clone. In some cases, the VL-FR2 sequence can be the same VL-FR2 sequence as the initial antibody clone. In some cases, the VL-FR3 sequence can be the same VL-FR3 sequence as the initial antibody clone. In some cases, the VL-FR4 sequence can be the same VL-FR4 sequence as the initial antibody clone.

[0135] The framework of the antibody may be a naturally occurring framework. A naturally occurring framework may be a framework found in a mammal. The mammal may be a primate, mouse, rat, pig, goat, rabbit, horse, cow, cat or dog. The primate may be a human. The framework may contain at least one variant compared to a naturally occurring framework. A variant may be a mutation, insertion or deletion. A variant may be a variant found in a nucleic acid sequence encoding the antibody or a variant found in an amino acid sequence of the antibody. Any suitable framework sequence may be used, such as those previously used in Phase I clinical trials ( Fig. 12A , Fig. 12BAs used herein, the framework of an antibody may refer to the framework regions of the variable heavy chain (VH-FR1, VH-FR2, VH-FR3, and VH-FR4), the framework regions of the variable light chain (VL-FR1, VL-FR2, VL-FR3, and VL-FR4), or a combination thereof. The framework regions of the antibodies in the antibody library may be the same as the germline framework regions.

[0136] The framework can be the best framework for treatment. The best framework for treatment can include at least one, at least two, at least three, at least four, at least five or all of the following properties selected from the group: a) safety previously demonstrated in human monoclonal antibodies, b) thermal stability; c) not prone to aggregation; d) including a single dominant allele at the amino acid level in the entire human population; e) including different typical topologies of CDRs; f) well expressed in bacteria; and g) well displayed on phages. The framework with safety previously demonstrated in human monoclonal antibodies can be the framework of antibodies that have been used in at least Phase I clinical trials. A thermally stable framework can be a framework that is stable at at least 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or above 100°C. A thermally stable framework can be a framework that can withstand a temperature increase of at least 3°C ​​per minute, 4°C per minute or 5°C per minute. A framework that expresses well in bacteria can be a framework that produces biologically active antibodies in bacteria. The bacteria can be Escherichia coli. The bacteria can be engineered bacteria. The bacteria can be bacteria optimized for antibody expression. A framework that is well displayed on a phage can be a framework that produces a biologically active antibody when displayed on the surface of a phage.

[0137] Examples of strategies for selecting frames are described in Fig.11 In which the ideal framework of the antibody can be an antibody that shows structural diversity, has been successfully used in Phase I clinical trials in humans, has low immunogenicity, shows aggregation resistance, shows adaptability, and is thermally stable. In some cases, if the antibody framework has an inherent autoreactivity to blood cells (e.g., IGHV4-34), has poor stability characteristics (e.g., IGHV2-5), has a V gene that is not found in at least 50% of individuals (e.g., IGHV4-b), shows a V gene that tends to aggregate (e.g., IGLV6-57), or a combination thereof, the antibody framework is avoided.

[0138] The amino acid sequences of the antibody frameworks herein may contain more than one dominant allele, wherein different dominant alleles exist in different human populations ( Fig.13 and Fig.14For example, the IGHV1-3 framework contains three alleles: IGVH1-3*01, IGVH1-3*02, and IGVH1-3*03, which are found at different frequencies in different human populations ( Fig.26 ). In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in at least two human populations. In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in all human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least two human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least twelve human populations. In some cases, the framework region of the VH domain is a framework region from IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15, or IGHV3-23. In some cases, the framework region of the VH domain is a framework region from IGHV2-5, IGHV3-7, IGVH4-34, IGHV5-51, IGHV1-24, IGHV2-26, IGHV3-72, IGHV3-74, IGHV3-9, IGHV3-30, IGHV3-33, IGHV3-53, IGHV3-66, IGHV4-30-4, IGHV4-31, IGHV4-59, IGHV4-61, or IGHV5-51. In some cases, the framework region of the VH domain of the antibodies in the antibody library is a framework region from IGHV1-46, IGHV3-23, or a combination thereof. In some cases, the framework regions of the VL domains of the antibodies in the antibody library are framework regions from IGKV1-39, IGKV2-28, IGKV3-15, IGKV4-1, IGKV1-5, IGKV1-12, IGKV1-13, IGKV3-11, IGKV3-20, or a combination thereof. In one example, a subset of antibodies in the antibody library may have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV1-39, while the remaining antibodies in the antibody library have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV2-28.

[0139] In some cases, disclosed herein are nucleic acid sequences encoding antibodies described herein. The nucleic acid sequence may be a DNA or RNA sequence. The nucleic acid may be inserted into a vector. The vector may be a bacteriophage. The bacteriophage may be a phagemid or a bacteriophage. The phagemid may be pMID21. The bacteriophage may be DY3F63, M13 phage, fd filamentous phage, T4 phage, T7 phage, or lambda phage. In some cases, the phagemid may be introduced into a microorganism in combination with a bacteriophage (e.g., a "helper" phage). The microorganism may be a filamentous bacterium. The filamentous bacterium may be Escherichia coli.

[0140] The antibody library described herein comprises a plurality of antibodies. The plurality of antibodies may be at least 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 , 8.0×10 10 9.0×10 10 or 10.0×10 10 The plurality of antibodies may be at least 1.0×10 11 The plurality of antibodies may be at least 7.6×10 10 Due to the high diversity of such libraries, they can be unique. For example, in any library herein, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the plurality of antibodies can be unique. In some cases, the library has more than 7.0×10 10 The invention relates to a method for preparing a plurality of antibodies, wherein at least 20% of the plurality of antibodies are unique. Relative to other antibodies in the antibody library, a unique antibody can differ by at least one nucleic acid or at least one amino acid residue.

[0141] In some cases, the antibody library comprises at least 1.0×10 5 A plurality of antibodies, wherein at least 80% of the plurality of antibodies are functional (e.g., with a K of less than 100 nM d bind to the desired antigen). In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 80% of the plurality of antibodies are functional.

[0142] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 85% of the plurality of antibodies are functional.

[0143] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 90% of the plurality of antibodies are functional.

[0144] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 95% of the plurality of antibodies are functional.

[0145] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 99% of the plurality of antibodies are functional.

[0146] In various aspects, the antibody library may have a high diversity in one or more CDR sequences. In some cases, the antibody library may have a high diversity in VH-CDR1 sequences. In some cases, the antibody library may have a high diversity in VH-CDR2 sequences. In some cases, the antibody library may have a high diversity in VH-CDR3 sequences. In some cases, the antibody library may have a high diversity in VL-CDR1 sequences. In some cases, the antibody library may have a high diversity in VL-CDR2 sequences. In some cases, the antibody library may have a high diversity in VL-CDR3 sequences. In some cases, the antibody library may have a high diversity in CDR sequences that are not derived from the initial antibody clone, and have low diversity or no diversity in CDR sequences derived from the initial antibody clone. In some cases, an antibody library with a high diversity may contain at least 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 In some cases, the antibody library can include high diversity in five of the six CDR sequences, for example, the antibody library can include high diversity in five CDR sequences selected from the following: VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence. In such a case, the remaining CDR sequences can have low diversity or no diversity. In some cases, the remaining CDR sequences of every kind of antibody are identical CDR sequences. In some cases, the remaining CDR sequences are derived from initial antibody clones.

[0147] In various aspects, at least one antibody in the antibody library may exhibit improvement in at least one property compared to the initial antibody clone. In some cases, at least one antibody in the antibody library may exhibit improvement in thermal stability (e.g., higher Tm) compared to the initial antibody clone. For example, at least one antibody in the antibody library can have a melting temperature (Tm) that is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or more than 50°C higher than the initial antibody clone. In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the antibodies in the antibody library may have a higher Tm than the initial antibody clone.

[0148] In some cases, at least one antibody in the antibody library may exhibit a greater affinity (e.g., a lower dissociation constant (K)) for the target epitope than the initial antibody clone. d )). For example, at least one antibody in the antibody library may have a dissociation constant (K) greater than that of the initial antibody clone. d ) has a K for the target epitope that is at least 5X, at least 10X, at least 20X, at least 30X, at least 40X, at least 50X, at least 60X, at least 70X, at least 80X, at least 90X, at least 100X, at least 200X, at least 300X, at least 400X, at least 500X, at least 600X, at least 700X, at least 800X, at least 900X, or at least 1000X lower. d In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% of the antibodies in the antibody library may have a lower K than the initial antibody clone. d .

[0149] In some cases, at least one antibody in the antibody library may exhibit increased species selectivity compared to the initial antibody clone. For example, at least one antibody in the antibody library may have a K of 1.004 or 1.003 than the initial antibody clone. d In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% of the antibodies in the antibody library may have a lower Kd for an epitope from a particular species than the initial antibody clone. d .

[0150] In some cases, at least one antibody in the antibody library can exhibit increased species cross-reactivity (e.g., across primate species) compared to the initial antibody clone. For example, at least one antibody in the antibody library can have a lower K for epitope A of species A (e.g., cynomolgus monkey). d , and may have a higher affinity for a similar epitope A' of species B (e.g., human). In some cases, at least one antibody in the antibody library may have a K greater than that of the initial antibody clone. d The K of the target epitope in the first species is at least 5X, at least 10X, at least 20X, at least 30X, at least 40X, at least 50X, at least 60X, at least 70X, at least 80X, at least 90X, at least 100X, at least 200X, at least 300X, at least 400X, at least 500X, at least 600X, at least 700X, at least 800X, at least 900X, or at least 1000X lower. d , and may also have a K higher than the initial antibody clone d The K of the epitope to a similar epitope in a second species is at least 5X, at least 10X, at least 20X, at least 30X, at least 40X, at least 50X, at least 60X, at least 70X, at least 80X, at least 90X, at least 100X, at least 200X, at least 300X, at least 400X, at least 500X, at least 600X, at least 700X, at least 800X, at least 900X, or at least 1000X lower. dIn some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the antibodies in the antibody library may have a lower K for an epitope from a first species and for a similar epitope from a second species compared to the initial antibody clone. d .

[0151] In various fields, antibody library can comprise and compare with initial antibody clone and show one or more antibodies improved in more than one characteristic.In some cases, this improvement is selected from: improved thermostability, improved affinity to target epitope, improved selectivity to target epitope of specific species and improved interspecies cross reactivity.In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or be greater than 95% in following two in show improvement: thermostability, affinity to target epitope, selectivity to target epitope of specific species or interspecies cross reactivity. In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% of the antibodies in the antibody library exhibit improvements in three of the following: thermal stability, affinity for the target epitope, selectivity for the target epitope for a particular species, or cross-reactivity between species. Fig.31A and Fig.31B Depicted are non-limiting examples of selecting antibody clones that exhibit improved thermal stability, improved affinity for Epitope A from Cynomolgus monkey, and improved affinity for Epitope A from human.

[0152] In various aspects, the antibodies in the antibody library can exhibit thermal stability. In some cases, the antibodies in the antibody library can have a melting temperature (Tm) between about 50°C to about 90°C. In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more than 95% of the antibodies in the antibody library have a melting temperature (Tm) between about 50°C to about 90°C. For example, the antibodies in the antibody library can have a melting temperature (Tm) of at least 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

[0153] In various aspects, the antibodies in the antibody library can show high affinity to the target epitope. In some cases, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more than 95% of the antibodies in the antibody library can show high affinity to the target epitope. For example, the antibodies in the antibody library can have a dissociation constant (K) of less than about 50 nM, 25 nM, 10 nM, 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100 fM, 50 fM, 25 fM, 10 fM, 5 fM, 1 fM, or less. d ) binds to the target epitope. Method for generating antibody libraries using Tumbler

[0154] In one aspect, a method for generating an antibody library (such as the above-mentioned antibody library) is provided. In some cases, the method comprises: (a) selecting a CDR sequence, wherein the CDR sequence is selected from: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; (b) replacing the CDR sequence of each antibody in the first antibody library with the CDR sequence selected in (a), thereby generating a second antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (i) the CDR sequence selected in (a); and (ii) a unique combination of the remaining CDR sequences not selected in (a), wherein the remaining CDR sequences are selected from: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.

[0155] Fig.32 and Fig.33 A non-limiting exemplary workflow of a method for generating an antibody library and obtaining one or more desired antibodies therefrom is depicted. In some cases, an initial antibody clone is obtained ( Fig.32 , 3201). In some cases, the initial antibody clones can be obtained from a third party (such as a customer or client). In other cases, the initial antibody clones can be obtained from a highly diverse antibody library (e.g., the SuperHuman antibody library described herein). In some cases, the initial antibody clones can have desired properties, such as affinity for a particular epitope. In some cases, it may be desirable to improve one or more properties of the initial antibody clones. For example, it may be desirable to improve the thermal stability of the antibody (e.g., increase the melting temperature (Tm)), the binding properties of the antibody (e.g., affinity), or the species cross-reactivity of the antibody between two or more species. The initial antibody clones can then be used to generate an antibody library ( Fig.32 , 3203). In some cases, a CDR sequence from an initial antibody clone can be selected. The CDR sequence can be any of the following: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, or a VL-CDR3 sequence. In a specific aspect, the CDR sequence is a VH-CDR3 sequence (see Fig.33). Typically, the CDR sequences selected from the initial antibody clones are CDR sequences that are important for the desired properties, such as CDR sequences that are important for binding affinity to the target epitope. In various aspects, the CDR sequences selected from the initial antibody clones can be cloned into a highly diverse antibody library. In some cases, the highly diverse antibody library can have high diversity in five of the six CDR sequences, but almost no diversity in one of the CDR sequences that is replaced (see, e.g., Fig.30 and Fig.33 In some cases, the highly diverse antibody library can be a SuperHuman antibody library or a modified SuperHuman antibody library (e.g., the same scaffold as the SuperHuman antibody library, but without diversity in the replaced CDR sequences; see Fig.33 ). In some cases, the CDR sequence of each antibody of the highly diverse antibody library can be replaced by a CDR sequence selected from the initial antibody clone, so that each antibody in the subsequent antibody library has the same CDR sequence. For example, the VH-CDR3 sequence from the initial antibody clone can be cloned into the highly diverse antibody library, so that each VH-CDR3 sequence in the highly diverse antibody library is replaced by the same VH-CDR3 sequence selected from the initial antibody clone (see Fig.33 ). In this case, the remaining CDR sequences (VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 in this example) are CDR sequences present in a highly diverse antibody library. In some cases, the CDR sequences can be cloned into a highly diverse antibody library using a method for introducing mutations into CDR sequences (e.g., to introduce more diversity into the CDR sequences). In some examples, the CDR sequences can be cloned by performing an error-prone PCR method to introduce one or more mutations into the CDR sequences. In some cases, each antibody in the highly diverse antibody library can have a unique combination of CDR sequences. Thus, such a method can generate an antibody library with high diversity in VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3, but little diversity in VH-CDR3 (see Fig.33 Additional selection and screening steps can be performed on subsequent antibody libraries to select antibody clones with desired properties ( Fig.32 , 3205). Finally, the optimal antibody sequence can be determined by computational methods ( Fig.32 , 3207). Fig.34 and Fig.35Described are methods of screening and selecting antibody clones having improved properties, such as increased thermal stability compared to the initial antibody clone, increased binding affinity to a target epitope compared to the initial antibody clone, and / or increased species cross-reactivity compared to the initial antibody clone.

[0156] In various aspects, a method for generating an antibody library is provided. In some cases, the antibody library may include multiple antibodies, wherein each antibody in the multiple antibodies includes: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the CDR sequence is selected from the following: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence, wherein the CDR sequence of each antibody in the multiple antibodies is the same; and (d) the unique combination of the remaining CDR sequences is selected from the following: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3. The antibody library may also be referred to as a Tumbler library herein.

[0157] In one example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR1 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VH-CDR1 sequence is derived from an initial antibody clone.

[0158] In another example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR2 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR1 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VH-CDR2 sequence is derived from an initial antibody clone.

[0159] In another example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VH-CDR3 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VH-CDR3 sequence is derived from an initial antibody clone.

[0160] In another example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR1 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR2 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VL-CDR1 sequence is derived from an initial antibody clone.

[0161] In another example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR2 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, and the VL-CDR3 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VL-CDR2 sequence is derived from an initial antibody clone.

[0162] In another example, a method for generating an antibody library comprising a plurality of antibodies is provided, wherein each antibody in the plurality of antibodies comprises: (a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and (b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; wherein (c) the VL-CDR3 sequence is identical for each antibody in the plurality of antibodies; and (d) the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, and the VL-CDR2 sequence are present in different combinations in each antibody in the plurality of antibodies. In some cases, the VL-CDR3 sequence is derived from an initial antibody clone.

[0163] In various aspects, one or more of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence are naturally occurring. In various aspects, each of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence is naturally occurring, but is present in each antibody in a non-naturally occurring combination. In various aspects, one or more of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence are naturally occurring in the human population or are derived from human CDR sequences. In various aspects, each of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, and VL-CDR3 sequence naturally occurs in the human population, or is derived from a human CDR sequence.

[0164] In various aspects, the antibodies of the library may comprise non-naturally occurring combinations of naturally occurring CDRs, such as combinations of CDRs derived from naturally occurring memory B cells and naive B cells, but their co-occurrence on the same antibody is not naturally occurring. For example, the non-naturally occurring combination of naturally occurring CDRs may comprise at least one CDR derived from a naive cell, while the remaining CDRs may be derived from a memory cell. For example, the non-naturally occurring combination of naturally occurring CDRs may comprise at least one CDR derived from a cell that is primarily a naive B cell source, while the remaining CDRs may be derived from a cell that is primarily a memory B cell source.

[0165] The non-naturally occurring combination of naturally occurring CDRs may include at least one CDR derived from a naive cell, while the remaining CDRs are derived from a memory cell. In some cases, at least VL-CDR1 is derived from a naive cell. In some cases, at least VL-CDR2 is derived from a naive cell. In some cases, at least VL-CDR3 is derived from a naive cell. In some cases, at least VH-CDR1 is derived from a naive cell. In some cases, at least VH-CDR2 is derived from a naive cell. In some cases, at least VH-CDR3 is derived from a naive cell.

[0166] The non-natural combination of naturally occurring CDR can include two, three, four or five CDRs derived from initial cells, and the remaining CDRs can be derived from memory cells. For example, two CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, three CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, four CDRs from the CDR in the lower group can be derived from initial cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, and the remaining CDRs can be derived from memory cells. In another example, five CDRs from the following group of CDRs can be derived from naive cells: VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3, while the remaining CDRs can be derived from memory cells.

[0167] In another non-limiting example of a non-naturally occurring combination, VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR2 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 may be derived from a memory cell. In another non-limiting example of a non-naturally occurring combination, VH-CDR3 and VL-CDR3 may be derived from a naive cell, while VH-CDR1, VH-CDR2, VL-CDR1, and VL-CDR2 may be derived from a memory cell.

[0168] The VH-CDR1 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR1 sequence derived from a naive B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR2 sequence derived from a naive B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell. The VH-CDR3 sequence derived from a naive B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a naive B cell.

[0169] The VL-CDR1 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR1 sequence derived from the initial B cell may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR2 sequence derived from the initial B cell may be a synthetic VL-CDR2 sequence. The VL-CDR2 sequence derived from the initial B cell may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from the initial B cell. The VL-CDR3 sequence derived from a naive B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell. The VL-CDR3 sequence derived from a naive B cell may be a synthetic VL-CDR3 sequence. The VL-CDR3 sequence derived from a naive B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a naive B cell.

[0170] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from a naive B cell. The naive B cell pool can be obtained from multiple individuals. The naive B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from naive B cells.

[0171] The VH-CDR1 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR1 sequence derived from a memory B cell may be a synthetic VH-CDR1 sequence. The VH-CDR1 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR1 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR2 sequence derived from a memory B cell may be a synthetic VH-CDR2 sequence. The VH-CDR2 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR2 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell. The VH-CDR3 sequence derived from a memory B cell may be a synthetic VH-CDR3 sequence. The VH-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VH-CDR3 sequence from a memory B cell.

[0172] The VL-CDR1 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR1 sequence derived from memory B cells may be a synthetic VL-CDR1 sequence. The VL-CDR1 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR1 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR2 sequence derived from memory B cells may contain 100% sequence homology with the naturally occurring VL-CDR2 sequence from memory B cells. The VL-CDR3 sequence derived from a memory B cell may contain at least 80%, 85%, 90%, 95% or 99% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell. The VL-CDR3 sequence derived from a memory B cell may contain 100% sequence homology with a naturally occurring VL-CDR3 sequence from a memory B cell.

[0173] The VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence, VL-CDR3 sequence or any combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The VH-CDR3 sequence, VL-CDR3 sequence or a combination thereof can be derived from sequence information obtained from a cell pool that is mainly derived from memory B cells. The memory B cell pool can be obtained from multiple individuals. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from memory B cells. The memory B cells may be CD27+ B cells. The memory B cell pool may contain less than 0.1%, 1%, 5%, 10%, 20% or 30% of cells that are not derived from CD27+ B cells.

[0174] The naive cell may be a naive B cell. The naive B cell may be a human naive B cell. The memory cell may be a memory B cell. The memory B cell may be a human memory B cell. In some cases, the naive B cell exhibits increased diversity of VH-CDR3 and VL-CDR3 sequences compared to the VH-CDR3 and VL-CDR3 sequences from the memory B cell ( Figure 1 ). The naive cells and memory cells can be obtained from a biological sample, such as blood, from an individual or multiple individuals. The naive cells and memory cells can be physically separated from the sample using markers specific to the naive cells or memory cells.

[0175] Markers can be used to identify, separate or sort B cells, naive B cells and memory B cells from biological samples. Examples of markers for identifying, separating or sorting B cells include, but are not limited to, CD19+. Examples of markers for identifying, separating or sorting naive B cells include, but are not limited to, CD19+, CD27-, IgD+, IgM+ and combinations thereof. Examples of markers for identifying, separating or sorting memory B cells include, but are not limited to, CD19+, CD27+ and combinations thereof. In some embodiments, CD27+ is used to sort memory B cells. Examples of markers for identifying, separating or sorting class-converted memory B cells include, but are not limited to, CD19+, CD27+, CD27+, IgD-, IgM- and combinations thereof. Examples of markers for identifying, separating or sorting non-converted or marginal zone memory B cells include, but are not limited to, CD19+, CD27+, IgD+, IgM+ and combinations thereof. In some cases, memory B cells can be identified, separated or sorted with the following markers: CD19+, CD27+, IgD-, IgM+ and combinations thereof. The naive cell from which VH-CDR3 is derived may be a CD27- / IgM+ B cell. The memory cell from which VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2 and VL-CDR3 are derived may be a CD27+ / IgG+ B cell.

[0176] The CDR sequences of antibodies can be CDR sequences found in naive B cells and memory B cells found in a single or multiple individuals. The individual can be a mammal. The mammal can be a human, a non-human primate, a mouse, a rat, a pig, a goat, a rabbit, a horse, a cow, a cat or a dog. In some cases, the CDR sequences are CDR sequences obtained from publicly available sources. Examples of sources of publicly available CDR sequences include SAbDab (http: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / Welcome.php) and PylgClassify (http: / / dunbrack2.fccc.edu / PyIgClassify / ).

[0177] In various aspects, each antibody in the antibody library can comprise the same scaffold, e.g., the same combination of framework sequences (see Fig.30). In various aspects, the VH domain of each antibody in the antibody library comprises a VH-FR1 sequence, a VH-FR2 sequence, a VH-FR3 sequence, and a VH-FR4 sequence. In some cases, each of the VH-FR1 sequence, the VH-FR2 sequence, the VH-FR3 sequence, and the VH-FR4 sequence of each antibody in the antibody library is identical. In some cases, each of the VH-FR1 sequence, the VH-FR2 sequence, the VH-FR3 sequence, and the VH-FR4 sequence is derived from the same initial antibody clone from which the CDR sequences are derived (see Fig.30 ). In some cases, the VH-FR1 sequence may be the same VH-FR1 sequence as the initial antibody clone. In some cases, the VH-FR2 sequence may be the same VH-FR2 sequence as the initial antibody clone. In some cases, the VH-FR3 sequence may be the same VH-FR3 sequence as the initial antibody clone. In some cases, the VH-FR4 sequence may be the same VH-FR4 sequence as the initial antibody clone. In various aspects, the VL domain of each antibody in the antibody library comprises a VL-FR1 sequence, a VL-FR2 sequence, a VL-FR3 sequence, and a VL-FR4 sequence. In some cases, each of the VL-FR1 sequence, the VL-FR2 sequence, the VL-FR3 sequence, and the VL-FR4 sequence of each antibody in the antibody library is the same. In some cases, each of the VL-FR1 sequence, the VL-FR2 sequence, the VL-FR3 sequence, and the VL-FR4 sequence may be derived from the same initial antibody clone from which the CDR sequence is derived (see Fig.30 ). In some cases, the VL-FR1 sequence can be the same VL-FR1 sequence as the initial antibody clone. In some cases, the VL-FR2 sequence can be the same VL-FR2 sequence as the initial antibody clone. In some cases, the VL-FR3 sequence can be the same VL-FR3 sequence as the initial antibody clone. In some cases, the VL-FR4 sequence can be the same VL-FR4 sequence as the initial antibody clone.

[0178] The framework of the antibody may be a naturally occurring framework. A naturally occurring framework may be a framework found in a mammal. The mammal may be a primate, mouse, rat, pig, goat, rabbit, horse, cow, cat or dog. The primate may be a human. The framework may contain at least one variant compared to a naturally occurring framework. A variant may be a mutation, insertion or deletion. A variant may be a variant found in a nucleic acid sequence encoding the antibody or a variant found in an amino acid sequence of the antibody. Any suitable framework sequence may be used, such as those previously used in Phase I clinical trials ( Fig. 12A , Fig. 12BAs used herein, the framework of an antibody may refer to the framework regions of the variable heavy chain (VH-FR1, VH-FR2, VH-FR3, and VH-FR4), the framework regions of the variable light chain (VL-FR1, VL-FR2, VL-FR3, and VL-FR4), or a combination thereof. The framework regions of the antibodies in the antibody library may be the same as the germline framework regions.

[0179] The framework can be the best framework for treatment. The best framework for treatment can include at least one, at least two, at least three, at least four, at least five or all of the following properties selected from the group: a) safety previously demonstrated in human monoclonal antibodies, b) thermal stability; c) not prone to aggregation; d) including a single dominant allele at the amino acid level in the entire human population; e) including different typical topologies of CDRs; f) well expressed in bacteria; and g) well displayed on phages. The framework with safety previously demonstrated in human monoclonal antibodies can be the framework of antibodies that have been used in at least Phase I clinical trials. A thermally stable framework can be a framework that is stable at at least 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C or above 100°C. A thermally stable framework can be a framework that can withstand a temperature increase of at least 3°C ​​per minute, 4°C per minute or 5°C per minute. A framework that expresses well in bacteria can be a framework that produces biologically active antibodies in bacteria. The bacteria can be Escherichia coli. The bacteria can be engineered bacteria. The bacteria can be bacteria optimized for antibody expression. A framework that is well displayed on a phage can be a framework that produces a biologically active antibody when displayed on the surface of a phage.

[0180] Examples of strategies for selecting frames are described in Fig.11 In which the ideal framework of the antibody can be an antibody that shows structural diversity, has been successfully used in Phase I clinical trials in humans, has low immunogenicity, shows aggregation resistance, shows adaptability, and is thermally stable. In some cases, if the antibody framework has an inherent autoreactivity to blood cells (e.g., IGHV4-34), has poor stability characteristics (e.g., IGHV2-5), has a V gene that is not found in at least 50% of individuals (e.g., IGHV4-b), shows a V gene that tends to aggregate (e.g., IGLV6-57), or a combination thereof, the antibody framework is avoided.

[0181] The amino acid sequences of the antibody frameworks herein may contain more than one dominant allele, wherein different dominant alleles exist in different human populations ( Fig.13 and Fig.14For example, the IGHV1-3 framework contains three alleles: IGVH1-3*01, IGVH1-3*02, and IGVH1-3*03, which are found at different frequencies in different human populations ( Fig.26 ). In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in at least two human populations. In some cases, the amino acid sequence of the antibody framework described herein has a single dominant allele in all human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least two human populations. A framework with a dominant allele can be a framework in which an allele is found in at least 50%, at least 75%, or at least 90% of at least twelve human populations. In some cases, the framework region of the VH domain is a framework region from IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15, or IGHV3-23. In some cases, the framework region of the VH domain is a framework region from IGHV2-5, IGHV3-7, IGVH4-34, IGHV5-51, IGHV1-24, IGHV2-26, IGHV3-72, IGHV3-74, IGHV3-9, IGHV3-30, IGHV3-33, IGHV3-53, IGHV3-66, IGHV4-30-4, IGHV4-31, IGHV4-59, IGHV4-61, or IGHV5-51. In some cases, the framework region of the VH domain of the antibodies in the antibody library is a framework region from IGHV1-46, IGHV3-23, or a combination thereof. In some cases, the framework regions of the VL domains of the antibodies in the antibody library are framework regions from IGKV1-39, IGKV2-28, IGKV3-15, IGKV4-1, IGKV1-5, IGKV1-12, IGKV1-13, IGKV3-11, IGKV3-20, or a combination thereof. In one example, a subset of antibodies in the antibody library may have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV1-39, while the remaining antibodies in the antibody library have a framework region from the VH domain of IGHV1-46 and a framework region from the VL domain of IGKV2-28.

[0182] In some cases, disclosed herein are nucleic acid sequences encoding antibodies described herein. The nucleic acid sequence may be a DNA or RNA sequence. The nucleic acid may be inserted into a vector. The vector may be a bacteriophage. The bacteriophage may be a phagemid or a bacteriophage. The phagemid may be pMID21. The bacteriophage may be DY3F63, M13 phage, fd filamentous phage, T4 phage, T7 phage, or lambda phage. In some cases, the phagemid may be combined with a bacteriophage (i.e., a "helper" phage) and introduced into a microorganism. The microorganism may be a filamentous bacterium. The filamentous bacterium may be Escherichia coli.

[0183] The antibody library described herein comprises a plurality of antibodies. The plurality of antibodies may be at least 1.0×10 6 , 1.0×10 7 , 1.0×10 8 , 1.0×10 9 , 1.0×10 10 , 2.0×10 10 , 3.0×10 10 4.0×10 10 , 5.0×10 10 , 6.0×10 10 , 7.0×10 10 , 8.0×10 10 9.0×10 10 or 10.0×10 10 The plurality of antibodies may be at least 1.0×10 11 The plurality of antibodies may be at least 7.6×10 10 Due to the high diversity of such libraries, they can be unique. For example, in any library herein, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% of the plurality of antibodies can be unique. In some cases, the library has more than 7.0×10 10 The invention relates to a method for preparing a plurality of antibodies, wherein at least 20% of the plurality of antibodies are unique. Relative to other antibodies in the antibody library, a unique antibody can differ by at least one nucleic acid or at least one amino acid residue.

[0184] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 80% of the plurality of antibodies are functional.

[0185] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 85% of the plurality of antibodies are functional.

[0186] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 90% of the plurality of antibodies are functional.

[0187] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 95% of the plurality of antibodies are functional.

[0188] In some cases, the antibody library comprises at least 1.0×10 5 In some cases, the antibody library comprises at least 7.0×10 10 In some cases, the antibody library comprises at least 7.6×10 10 The method further comprises the step of: obtaining a plurality of antibodies, wherein at least 99% of the plurality of antibodies are functional.

[0189] In various aspects, the methods provided herein can generate antibody libraries with high diversity in one or more CDR sequences. In some cases, the antibody library can have high diversity in the VH-CDR1 sequence. In some cases, the antibody library can have high diversity in the VH-CDR2 sequence. In some cases, the antibody library can have high diversity in the VH-CDR3 sequence. In some cases, the antibody library can have high diversity in the VL-CDR1 sequence. In some cases, the antibody library can have high diversity in the VL-CDR2 sequence. In some cases, the antibody library can have high diversity in the VL-CDR3 sequence. In some cases, the antibody library can have high diversity in the CDR sequence that is not derived from the initial antibody clone, and has low diversity or no diversity in the CDR sequence derived from the initial antibody clone. In some cases, the antibody library with high diversity can contain at least 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 In some cases, the antibody library can include high diversity in five of the six CDR sequences, for example, the antibody library can include high diversity in five CDR sequences selected from the following: VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence. In such a case, the remaining CDR sequences can have low diversity or no diversity. In some cases, the remaining CDR sequences of every kind of antibody are identical CDR sequences. In some cases, the remaining CDR sequences are derived from initial antibody clones.

[0190] In various aspects, compared with the initial antibody clone, the method provided herein can generate at least one antibody with improvement in at least one characteristic. In some cases, compared with the initial antibody clone, at least one antibody in the antibody library can show an improvement in thermal stability. For example, compared with the initial antibody clone, at least one antibody in the antibody library can show at least 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 55X, 60X, 65X, 70X, 75X, 80X, 85X, 90X, 95X, 100X or thermal stability greater than 100X. In some cases, compared with the initial antibody clone, at least one antibody in the antibody library can show a greater affinity to the target epitope. For example, at least one antibody in the antibody library can exhibit an affinity for the target epitope that is at least 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 55X, 60X, 65X, 70X, 75X, 80X, 85X, 90X, 95X, 100X, or greater than 100X as compared to the initial antibody clone. In some cases, at least one antibody in the antibody library can exhibit an improvement in species selectivity. For example, at least one antibody in the antibody library can exhibit an affinity for a target epitope of a particular species that is at least 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 55X, 60X, 65X, 70X, 75X, 80X, 85X, 90X, 95X, 100X, or greater than 100X compared to the initial antibody clone. In some cases, at least one antibody in the antibody library can exhibit increased species cross-reactivity compared to the initial antibody clone. For example, at least one antibody in the antibody library can have a high affinity for epitope A from species A (e.g., cynomolgus monkey) and can have a high affinity for epitope A from species B (e.g., humans).In some cases, at least one antibody in the antibody library may exhibit at least 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 55X, 60X, 65X, 70X, 75X, 80X, 85X, 90X, 95X, 100X, or more than 100X of affinity for antibodies from species as compared to the initial antibody clone. The antibody clones described herein may further comprise an antibody that has an affinity for epitope A from species B and exhibits an affinity for epitope A from species B that is at least 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 55X, 60X, 65X, 70X, 75X, 80X, 85X, 90X, 95X, 100X, or greater than 100X the affinity for epitope A from species B as compared to the initial antibody clone. Fig.31A and Fig.31B Depicted are non-limiting examples of selecting antibody clones that exhibit improved thermal stability, improved affinity for Epitope A from Cynomolgus monkey, and improved affinity for Epitope A from human.

[0191] In various aspects, the methods described herein can generate antibodies with thermal stability. In some cases, the antibodies in the antibody library can be thermally stable at a temperature of about 50° C. to about 90° C. For example, the antibodies in the antibody library can be thermally stable at a temperature of at least 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., or 90° C.

[0192] In various aspects, the methods described herein can generate antibodies with high affinity for a target epitope. For example, the antibodies in the antibody library can have a dissociation constant (K) of less than about 50 nM, 25 nM, 10 nM, 5 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100 fM, 50 fM, 25 fM, 10 fM, 5 fM, 1 fM, or less. d ) binds to the target epitope. Certain terms

[0193] The terms used herein are only for the purpose of describing a particular situation and are not intended to be limiting. In addition to the understanding of these terms by those skilled in the art, the following terms are discussed to illustrate the meaning of the terms used in this specification. As used herein and in the appended claims, unless the context clearly provides otherwise, the singular forms "one", "a kind of" and "the" include plural referents. It should also be noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to be used as an exclusive term such as "alone", "only" or the prior basis for using a "negative" restriction in association with the narration of the claimed element.

[0194] Certain ranges are given herein, wherein the value is preceded by the term "about". The term "about" is used herein to provide textual support for the exact number that follows and the number that is close to or approximate to the number following the term. When determining whether a number is close to or approximate to a specifically listed number, the close or approximate unlisted number can be such a number that, in the context of the given number, provides a substantially equivalent form of the specifically listed number. In the case of providing a numerical range, it is understood that, unless the context clearly specifies otherwise, each intermediate value between the upper and lower limits of the range and any other described value or intermediate value within the range are included in the methods and compositions described herein on one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also included in the methods and compositions described herein, but are subject to any explicitly excluded limitations within the range. When the range includes one or two limit values, the range excluding one or two of those included limit values ​​is also included in the methods and compositions described herein.

[0195] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, nursing assistant, or hospice worker). Additionally, these terms refer to either a human or animal subject.

[0196] "Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathological condition or disorder. Individuals in need of treatment include those already with the disorder as well as those susceptible to having the disorder or those in whom the disorder is to be prevented.

[0197] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as various forms including full-length antibodies and portions thereof; including, for example, immunoglobulin molecules, polyclonal antibodies, monoclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, F(ab)2, Fv, scFv, IgGΔCH2, F(ab')2, scFv2CH3, F(ab), VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, disulfide-linked Fv, single domain antibodies (dAb), diabodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, bispecific antibodies, any isotype (including but not limited to IgA, IgD, IgE, IgG or IgM), modified antibodies and synthetic antibodies (including but not limited to non-depleting IgG antibodies, T antibodies or other Fc or Fab variants of antibodies).

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the methods and compositions described herein belong. Although any methods and materials similar or equivalent to those described herein can also be used to implement or test the methods and compositions described herein, representative exemplary methods and materials are now described. Example

[0199] Example 1: Generation of SuperHuman Library (SHL) 2.0 Generate the SuperHuman library using the following steps: 1. The best 4 VH frameworks and the best 4 VK frameworks from the human repertoire of 3500 combinations (IGHV1-46, IGHV1-69, IGHV3-15, IGHV3-23 for heavy chain, and IGKV1-39, IGKV2-28, IGKV3-15, IGKV4-1 for light chain) were selected based on the following combination: 1) safety previously demonstrated in human mAbs, 2) thermal stability; 3) no tendency to aggregation; 4) a single dominant allele in the framework at the amino acid level in all human populations (i.e., non-ethnic drug); 5) different canonical topologies of the CDRs; 6) good expression in bacteria and good display on phage.

[0200] 2. Blood was obtained from 140 subjects.

[0201] 3. Isolate naive (CD27- / IgM+) cells and memory (CD27+ / IgG+) cells from blood.

[0202] 4. Check the quality of the pools using next generation sequencing (NGS) and discard pools with problematic diversity or biochemical tropism.

[0203] 5. PCR amplification of the VH-CDR3 sequence from the initial cells using universal primers.

[0204] 6. PCR amplify the VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2 and VL-CDR3 sequences from memory cells using framework-specific primers.

[0205] 7. Framed to synthetically generated germline segments.

[0206] 8. Assemble the nucleic acid library using PCR-OE.

[0207] 9. Check the quality of the assembly from step 8 using NGS sequencing.

[0208] 10. Clone the light chain into the vector with filled VH.

[0209] 11. Select the in-frame material by using Protein A or Protein L after heat stress.

[0210] 12. Clone the heavy chain into the vector to replace the filler VH.

[0211] 13. Transform the microorganism with the vector generated at the end of step 12 using electroporation.

[0212] Example 2: Screening for affinity to PD1 Initial screening of clones from two randomly selected 96-well plates after the fourth round of SuperHuman panning for PD1.

[0213] Bypass ELISA screening and immediately assay samples on a Carterra high-throughput kinetic instrument ( Figure 3 ). The majority of samples (hits) were positive, and 98 out of 184 sequences were unique.

[0214] Clones showing affinity for PD1 were demonstrated for both human and cynomolgus monkey PD1 ( Figure 4 )、( Figure 5 ).

[0215] Example 3: bGal ELISA and Sanger screening of two plates of antibody clones Antibody clones from both plates were subjected to ELISA panning against bGal ( Figure 2).

[0216] These clones were also subjected to Sanger sequencing ( Figure 8 The extreme diversity of the third-round output ensures that the number of clones sampled for any epitope can be recovered by screening a few 96-well plates of clones.

[0217] Diversity was found not only in the VH-CDR3 (CDR-H3) sequences, but also in the VH-CDR1 (CDR-H1) and VH-CDR2 (CDR-H2) sequences ( Fig.10 ).

[0218] Example 4: Combinatorial design and selection methods to generate antibody libraries with diverse VH and VK sequences Functional selection for expression and thermostability was applied during construction to generate a library with more than 95% functional diversity among 40 million light chains. 10 The antibody library was constructed from each transformant.

[0219] First, a VK (kappa light chain) library is generated by cloning the desired light chain and a temporary stuffer VH sequence into a vector. The VK library is displayed and subjected to heat stress at over 65°C. In-frame material is selected using protein A / L. The stuffer VH sequence in the library generated by protein A / L selection is replaced with the target VH sequence ( Fig.17 ).

[0220] Example 5: Generation of SuperHuman Library (SHL) 3.0 Generate the SuperHuman library using the following steps: 1. Six antibody frameworks (IGHV1-46, IGHV3-23, IGKV1-39, IGKV2-28, IGKV3-15, and IGKV4-1) were selected based on a combination of: 1) previously demonstrated safety in human mAbs, 2) thermal stability, 3) lack of aggregation susceptibility, 4) a single dominant allele in the framework at the amino acid level across all human populations (i.e., non-ethnic drug), 5) different canonical topologies of the CDRs, and 6) good expression in bacteria and good display on phage.

[0221] 2. Obtain blood from 50-100 subjects.

[0222] 3. Isolate naive (CD27- / IgM+ or CD27- / IgD+) cells and memory cells from blood.

[0223] 4. Check the quality of the pools using next generation sequencing (NGS) and discard pools with problematic diversity or biochemical tropism.

[0224] 5. PCR amplification of the VH-CDR3 sequence from the initial cells using universal primers.

[0225] 6. Select unbiased favorable VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 sequences by DNA synthesis based on the following: (1) observed to exist in natural human antibodies, (2) observed not to perform poorly in SuperHuman2.0 selections against multiple antigens, (3) no biochemical bias (C, exposed M, deamination site, acid hydrolysis site, N-linked glycosylation site, amber stop codon, opal stop codon, high positive charge), and (4) mutations do not exceed a threshold (e.g., no more than 3 amino acid mutations per CDR). In other words, VH-CDR1, VH-CDR2, VL-CDR1, VL-CDR2, and VL-CDR3 sequences are synthesized if they meet the following criteria:

[0226] a) for each framework used, has no more than 4 amino acid mutations compared to the respective germline CDR; and b) was identified during NGS as being present in at least 2 subjects and was enriched without fitness disadvantage when evaluating a pool of 55,000 hits for 11 antigens from SuperHuman 2.0 (Example 1), or was not observed in humans but was sufficiently enriched when panning in the same SuperHuman 2.0 pool; and c) does not contain any biochemical propensities (N-linked glycosylation, deamination, acid hydrolysis, positively charged internal peptide cleavage, free cysteine, free methionine, alternative stop codons, cryptic splice sites, TEV cleavage sites, or excessively positively charged CDRs).

[0227] 7. Frameworks were set to synthetically generated 100% germline segments without mutations.

[0228] 8. Assemble the nucleic acid library using PCR-OE or another DNA assembly method.

[0229] 9. Check the quality of the assembly from step 8 using NGS sequencing.

[0230] 10. Clone the light chain into the vector with the stuffed VH 11. Select the in-frame material by using Protein A or Protein L after heat stress.

[0231] 12. Clone the heavy chain into the vector to replace the filler VH.

[0232] 13. Transform the microorganism with the vector generated at the end of step 12 using electroporation.

[0233] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to implement the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

[0234] This article also provides the following items: 1. An antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and b) a VL structure comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; in: a) at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from a naive B cell; b) if only one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the naive B cell, the VH-CDR3 sequence or the VL-CDR3 sequence that is not derived from the naive B cell is derived from a memory cell; and c) the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence are derived from memory B cells.

[0235] 2. The antibody library according to item 1, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from a naive B cell is a naturally occurring sequence.

[0236] 3. The antibody library according to item 1, wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from memory cells is a naturally occurring sequence.

[0237] 4. The antibody library according to item 1, wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells are naturally occurring sequences.

[0238] 5. The antibody library according to item 1, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from the naive B cell comprises at least 80% sequence homology with a naturally occurring sequence.

[0239] 6. The antibody library according to item 1, wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from memory cells contains at least 80% sequence homology with a naturally occurring sequence.

[0240] 7. The antibody library according to item 1, wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells contain at least 80% sequence homology with naturally occurring sequences.

[0241] 8. The antibody library according to item 1, wherein the VL domain is a VK domain or a Vλ domain.

[0242] 9. The antibody library according to item 1, wherein the initial B cells are CD27- / IgM+ B cells or CD27- / IgD+ B cells.

[0243] 10. The antibody library according to item 1, wherein the memory B cells are selected from CD27+ / IgG+ B cells, CD27+ / IgM+ B cells, IgA+ B cells and combinations thereof.

[0244] 11. The antibody library according to item 1, wherein the naive B cells and memory B cells are from a sample comprising a plurality of naive B cells and memory B cells sampled from a plurality of individuals.

[0245] 12. The antibody library according to item 11, wherein the plurality of individuals is at least 50 individuals.

[0246] 13. The antibody library according to item 1, wherein the plurality of antibodies are expressed on the surface of a plurality of bacteriophages.

[0247] 14. The antibody library according to item 13, wherein the plurality of bacteriophages are bacterial phages or phagemids.

[0248] 15. The antibody library according to item 13, wherein each phage in the plurality of phages comprises a nucleic acid sequence encoding: i) an antibody in the plurality of antibodies, and ii) a gene encoding a phage coat protein.

[0249] 16. The antibody library according to item 15, wherein the bacteriophage coat protein is protein gIII.

[0250] 17. The antibody library according to item 15, wherein the expression of the nucleic acid sequence of each bacteriophage produces an antibody fused to a bacteriophage coat protein.

[0251] 18. The antibody library according to item 1, wherein the VH domain further comprises a framework region selected from the group consisting of IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15 and IGHV3-23.

[0252] 19. The antibody library according to item 1, wherein the VL domain further comprises a framework region selected from the group consisting of IGKV1-39, IGKV2-28, IGKV3-15 and IGKV4-1.

[0253] 20. The antibody library according to item 1, wherein the plurality of antibodies comprises at least 7.6×10 10 Antibodies.

[0254] 21. The antibody library according to item 1, wherein at least 95% of the plurality of antibodies are functional.

[0255] 22. A method for preparing an antibody library, comprising: a) obtaining sequence information of multiple VH-CDR3 and VL-CDR3 sequences from the naive B cell pool, and obtaining sequence information of multiple VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences from the memory B cell pool; b) assembling a plurality of variable light (VL) domain sequences, each VL domain sequence comprising: a VL-CDR1 sequence obtained from the sequence information from the memory B cell determined in step a, a VL-CDR2 sequence obtained from the sequence information from the memory B cell determined in step a, and a VL-CDR3 sequence obtained from the sequence information from the memory B cell or the naive B cell determined in step a; c) assembling a plurality of first nucleic acid sequences encoding a plurality of first antibodies, each first antibody comprising: i. the variable light (VL) domain sequence assembled in step b; and ii. a single fixed heavy chain sequence; d) inserting the plurality of first nucleic acid sequences into a plurality of bacteriophages; e) expressing the plurality of first antibodies on the surface of the plurality of phages; f) applying at least one selection pressure to the plurality of phage to produce a subset of phage comprising a subset of the first nucleic acid sequences; g) assembling a plurality of variable heavy (VH) domain sequences, each VH domain sequence comprising: a VH-CDR1 sequence obtained from the sequence information from the memory B cell determined in step a, a VH-CDR2 sequence obtained from the sequence information from the memory B cell determined in step a, and a VH-CDR3 sequence obtained from the sequence information from the memory B cell or the naive B cell determined in step a, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the sequence information from the naive B cell; h) replacing said single fixed heavy chain sequence from a subset of said first nucleic acid sequences with said plurality of VH domain sequences assembled in step g to generate a plurality of second nucleic acid sequences, each second nucleic acid sequence comprising: i. the variable light (VL) domain sequence assembled in step b, and ii. the variable heavy (VH) domain sequence assembled in step g, wherein the plurality of second nucleic acid sequences encode a plurality of second antibodies; i) transforming a plurality of microorganisms using the plurality of bacteriophages to produce a plurality of transformants.

[0256] 23. The method according to item 22, wherein the naive B cell pool comprises less than 5% of cells that are not derived from naive B cells.

[0257] 24. The method according to item 22, wherein the memory B cell pool comprises less than 5% of cells that are not derived from memory B cells.

[0258] 25. The method according to item 1, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from a naive B cell is a naturally occurring sequence.

[0259] 26. The method according to item 1, wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from a memory cell is a naturally occurring sequence.

[0260] 27. The method according to item 1, wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells are naturally occurring sequences.

[0261] 28. The method according to item 1, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from the naive B cell comprises at least 80% sequence homology with a naturally occurring sequence.

[0262] 29. The method according to item 1, wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from a memory cell comprises at least 80% sequence homology with a naturally occurring sequence.

[0263] 30. The method according to item 1, wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells contain at least 80% sequence homology with naturally occurring sequences.

[0264] 31. The method according to item 22, wherein the naive B cell pool, the memory cell pool or a combination thereof is obtained from a plurality of individuals.

[0265] 32. The method of item 25, wherein the plurality of individuals is at least 50 individuals.

[0266] 33. The method according to item 23, further comprising, before obtaining the sequence information, sorting the naive B cells and the memory B cells in the sample to produce the naive B cell pool and the memory B cell pool.

[0267] 34. The method according to item 33, wherein sorting the naive B cells and the memory B cells comprises using flow cytometry.

[0268] 35. The method according to item 34, wherein the flow cytometry is fluorescence activated cell sorting (FACS).

[0269] 36. The method according to item 23, wherein the method further comprises extracting nucleic acid from the naive B cells and the memory B cells.

[0270] 37. The method according to item 36, wherein the nucleic acid is DNA.

[0271] 38. The method according to item 36, wherein the nucleic acid is mRNA.

[0272] 39. The method according to item 38, further comprising reverse transcribing the mRNA into complementary DNA (cDNA).

[0273] 40. The method of item 22, wherein assembling each VL domain sequence comprises using overlap extension PCR (OE-PCR).

[0274] 41. The method of item 22, wherein assembling each VH domain sequence comprises using overlap extension PCR (OE-PCR).

[0275] 42. The method according to item 22, wherein the single fixed heavy chain sequence is a germline sequence selected from the group consisting of IGHJ4, IGHV1-46, IGHV1-69, IGHV3-15 and IGHV3-23.

[0276] 43. The method according to item 22, wherein applying at least one selection pressure comprises applying heat stress, selecting with protein A, selecting with protein L, or a combination thereof.

[0277] 44. The method according to item 43, wherein the thermal stress is a temperature of at least 65°C.

[0278] 45. The method according to item 43, wherein applying thermal stress to the plurality of phages eliminates unstable and aggregation-prone phages from the phage subset.

[0279] 46. ​​The method according to item 43, wherein applying selection with protein A or protein L to the plurality of phages excludes phages expressing antibodies having no ability to bind to a protein from the phage subset.

[0280] 47. The method according to item 22, wherein the bacteriophage is a bacteriophage or a phagemid.

[0281] 48. The method according to item 22, wherein the microorganism is Escherichia coli.

[0282] 49. The method according to item 22, wherein the transformation is performed by electroporation.

[0283] 50. The method according to item 22, wherein the plurality of transformants comprises at least 7.6×10 10 A transformant.

[0284] 51. The method of item 22, the antibody library of item 1, wherein at least 95% of the plurality of antibodies are functional.

[0285] 52. An antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence and a VH-CDR3 sequence; and b) a VL domain comprising a VL-CDR1 sequence, a VL-CDR2 sequence and a VL-CDR3 sequence; wherein c) a CDR sequence is selected from the group consisting of a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence, wherein the CDR sequence is the same for each antibody in the plurality of antibodies; and d) a unique combination of the remaining CDR sequences is selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.

[0286] 53. The antibody library according to item 52, wherein the CDR sequence of (c) is a VH-CDR3 sequence.

[0287] 54. The antibody library according to item 53, wherein the remaining CDR sequences of (d) are VH-CDR1 sequences, VH-CDR2 sequences, VL-CDR1 sequences, VL-CDR2 sequences and VL-CDR3 sequences.

[0288] 55. The antibody library according to item 52, wherein the CDR sequence of (c) is identical to the CDR sequence derived from the initial antibody clone.

[0289] 56. The antibody library according to item 52, wherein each of the remaining CDR sequences of (d) is present in the antibody library with high diversity.

[0290] 57. The antibody library according to item 56, wherein the high diversity comprises at least 1×10 3 Different CDR sequences.

[0291] 58. The antibody library according to item 52, wherein at least one of the VH-CDR1 sequence, the VH-CDR2 sequence, the VH-CDR3 sequence, the VL-CDR1 sequence, the VL-CDR2 sequence and the VL-CDR3 sequence comprises at least 80% sequence homology with a naturally occurring CDR sequence.

[0292] 59. The antibody library according to item 58, wherein the naturally occurring CDR sequences are derived from the human population.

[0293] 60. The antibody library according to item 58, wherein the remaining CDR sequences of (d) are present in a non-naturally occurring combination for each antibody in the plurality of antibodies.

[0294] 61. The antibody library according to item 55, wherein at least one antibody in the plurality of antibodies has at least one of the following: a higher melting temperature (Tm) compared to the initial antibody clone, a higher affinity for a target epitope compared to the initial antibody clone, or a higher cross-reactivity to a target epitope between two or more species compared to the initial antibody clone.

[0295] 62. The antibody library according to item 52, wherein at least one antibody of the plurality of antibodies has a melting temperature (Tm) of about 50°C to about 90°C.

[0296] 63. The antibody library according to item 52, wherein at least one antibody of the plurality of antibodies has a K of 100 nM or less. d Binds to target epitope.

[0297] 64. A method for generating an antibody library, the method comprising: (a) selecting a CDR sequence, wherein the CDR sequence is selected from the group consisting of a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; (b) replacing the CDR sequence of each antibody in the first antibody library with the CDR sequence selected in (a), thereby generating a second antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: (i) the CDR sequences selected in (a); and (ii) a unique combination of remaining CDR sequences not selected in (a), wherein the remaining CDR sequences are selected from the group consisting of: a VH-CDR1 sequence, a VH-CDR2 sequence, a VH-CDR3 sequence, a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence.

[0298] 65. The method according to item 64, wherein the first antibody library comprises a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises a unique combination of VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence.

[0299] 66. The method according to item 64, wherein the CDR sequence selected in (a) is a VH-CDR3 sequence.

[0300] 67. The method according to item 66, wherein the remaining CDR sequences of ii) are VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence.

[0301] 68. The method according to item 64, wherein each of the remaining CDR sequences of ii) is present in the antibody library with high diversity.

[0302] 69. The method according to item 68, wherein the high diversity comprises at least 1×10 3Different CDR sequences.

[0303] 70. The method according to item 64, wherein at least one of the VH-CDR1 sequence, VH-CDR2 sequence, VH-CDR3 sequence, VL-CDR1 sequence, VL-CDR2 sequence and VL-CDR3 sequence in the antibody library comprises at least 80% sequence homology with a naturally occurring CDR sequence.

[0304] 71. The method according to item 70, wherein the naturally occurring CDR sequences are derived from the human population.

[0305] 72. The method according to item 64, wherein the remaining CDR sequences of ii) are present in a non-naturally occurring combination for each antibody in the plurality of antibodies.

[0306] 73. The method according to item 64, wherein the CDR sequences of (a) are derived from primary antibody clones.

[0307] 74. The method according to item 64, wherein at least one antibody in the antibody library has at least one of: a higher melting temperature (Tm) compared to the initial antibody clone, a higher affinity for the target epitope compared to the initial antibody clone, or a higher cross-reactivity to the target epitope between two or more species compared to the initial antibody clone.

[0308] 75. The method of item 64, wherein at least one antibody in the antibody library has a melting temperature (Tm) of about 50°C to about 90°C.

[0309] 76. The method of item 64, wherein at least one antibody in the antibody library has a K of 100 nM or less. d Binds to target epitope.

[0310] 77. The method according to item 64, wherein the first antibody library is the antibody library according to any one of items 1-21.

[0311] 78. The method according to item 64, wherein the second antibody library is an antibody library according to any one of items 52-63.

[0312] 79. The method according to item 64, further comprising (c) screening the second antibody library for antibodies having desired properties.

Claims

1. An antibody library comprising a plurality of antibodies, wherein each antibody in the plurality of antibodies comprises: a) a VH domain comprising a VH-CDR1 sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and b) a VL structure comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; in: a) at least one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from a naive B cell; b) if only one of the VH-CDR3 sequence and the VL-CDR3 sequence is derived from the naive B cell, the VH-CDR3 sequence or the VL-CDR3 sequence that is not derived from the naive B cell is derived from a memory cell; and c) the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence are derived from memory B cells. 2 . The antibody library according to claim 1 , wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from a naive B cell is a naturally occurring sequence. 3 . The antibody library according to claim 1 , wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from a memory cell is a naturally occurring sequence. 4 . The antibody library according to claim 1 , wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells are naturally occurring sequences.

5. The antibody library of claim 1, wherein at least one of the VH-CDR3 sequence and the VL-CDR3 sequence derived from a naive B cell comprises at least 80% sequence homology with a naturally occurring sequence.

6. The antibody library of claim 1, wherein the VH-CDR3 sequence or VL-CDR3 sequence derived from a memory cell comprises at least 80% sequence homology with a naturally occurring sequence.

7. The antibody library of claim 1, wherein the VH-CDR1 sequence, VH-CDR2 sequence, VL-CDR1 sequence and VL-CDR2 sequence derived from memory B cells comprise at least 80% sequence homology with naturally occurring sequences. The antibody library of claim 1 , wherein the VL domain is a VK domain or a Vλ domain.

9. The antibody library according to claim 1, wherein the naive B cells are CD27- / IgM+ B cells or CD27- / IgD+ B cells.

10. The antibody library of claim 1, wherein the memory B cells are selected from the group consisting of CD27+ / IgG+ B cells, CD27+ / IgM+ B cells, IgA+ B cells, and combinations thereof.

Images