阅读说明：本技术 犬抗体文库 (Canine antibody library ) 是由 T·蒂勒 M·瓦尔德胡贝尔 R·斯特罗纳 K·拉德茨基贝斯 J·普拉斯勒 于 2018-06-21 设计创作，主要内容包括：本发明提供合成犬抗体文库以及结合这些文库使用的多肽、核酸、载体、宿主细胞和方法。本发明还提供从这些文库分离的抗体。(The invention provides synthetic canine antibody libraries, as well as polypeptides, nucleic acids, vectors, host cells, and methods for use in conjunction with these libraries. The invention also provides antibodies isolated from these libraries.)

1. A synthetic canine antibody library, wherein the library comprises members of at least one of the following germline VH1 regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.: 2).

2. The library of any one of the preceding claims, wherein the library comprises at least one of the following germline VL-regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

3. The library of any one of the preceding claims, wherein post-translational modification (PTM) sites are removed from the germline VH region or one or more of the germline VL regions.

4. The library of any one of the preceding claims, wherein the library comprises VH regions: vs618(SEQ ID No.:4), Vs 624-PTM-minus (SEQ ID No.:6) and Vs 635-PTM-minus (SEQ ID No.:7), and includes a VL region: vs236(κ) (SEQ ID No.:12), Vs 323-PTM-Low (λ) (SEQ ID No.:18) and Vs365(λ) (SEQ ID No.: 13).

5. The library of any one of the preceding claims, wherein the library comprises VH regions: vs618(SEQ ID No.:4), Vs 624-PTM-low (SEQ ID No.:6), Vs 628-PTM-low (SEQ ID No.:10) and Vs 635-PTM-low (SEQ ID No.:7) and includes a VL region: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs 323-PTM-minus (λ) (SEQ ID No.:18), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

6. The library of any one of the preceding claims, wherein the library comprises one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs-843 (λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 6318), VH/VL combination of VH/VL region Vs 1-PTM-less (V4) and VL combination of VH region V-VL region 321, VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region VS236 (kappa) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 8415) VH/VL combinations.

7. The library of any one of the preceding claims, wherein substantially all VH/VL combinations of the library are effectively displayed at a display rate of at least 0.5 Fab/phage.

8. The library of any one of the preceding claims, wherein substantially all of the VH/VL combination has a monomer content of at least 85% when expressed in Fab form in e.

9. The library of any one of the preceding claims, wherein substantially all VH/VL combinations have a monomer content of at least 90% when expressed in an IgG format in a mammalian system.

10. The library of any one of the preceding claims, wherein all VH/VL combinations are thermostable.

11. A collection of nucleic acid molecules encoding members of the library of any one of the preceding claims.

12. A vector encoding the nucleic acid molecule of claim 11.

13. A recombinant host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method of isolating an antibody specific for an antigen, the method comprising the steps of:

(a) contacting the library of any one of claims 1-10 with an antigen;

(b) removing members of the library that do not bind to the antigen; and

(c) recovering the members of the library that bind to the antigen.

15. An antibody isolated from the library of any one of claims 1-10 or by the method of claim 14.

Technical Field

The present invention relates to and provides canine libraries, such as synthetic antibody libraries suitable for selecting fully canine antibodies. The invention also relates to synthetic nucleic acid sequences encoding individual or collective canine antibodies, i.e., nucleic acid sequences encoding a canine antibody library. Methods for generating and using such libraries are provided. In particular, the invention relates to the preparation of complete canine antibody libraries by using synthetic germline sequences to generate libraries of complete canine antibodies with specific biophysical properties.

Background

Immunoglobulins, such as antibodies, have a continuing and increasing interest in the pharmaceutical industry. Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially, and in 2007, 8 of the 20 best-selling biotech drugs in the united states were therapeutic monoclonal antibodies, each of which sold over 50 billion dollars annually throughout the world. Therapeutic antibodies improve the treatment of many diseases and increasingly improve the quality of life of patients, even the most severe and challenging of the disease.

Companion animals such as dogs develop similar diseases compared to humans, which are based on similar or even identical biological mechanisms and disorders. For example, lymphoma is the most common tumor of the canine blood lymphatic system. It represents about 4.5% of all canine tumors and 15% of all malignancies. Canine Lymphoma (CL) is often rapidly fatal, causing death within 1-3 months of diagnosis (Squire et al, 1973; Steven E.Crow, 2008). Furthermore, it is estimated that one fifth of the adult dogs in the united states have arthritis, and that dogs have been used as models of human joint disease, for example, for osteoarthritis, anterior cruciate ligament destruction, and meniscal damage.

Thus, therapeutic monoclonal antibodies provide a very promising class of drugs for the treatment of not only humans, but also dogs.

Attempts have been made to use antibodies for treatment of dogs. Even the first monoclonal antibody for human cancer therapy was approved by the U.S. food and Drug Administration, FDA, monoclonal antibody MAb231 for lymphoma-bearing dogs was approved by the United States Department of Agriculture (USDA) in 1992. MAb231 is a murine-derived monoclonal antibody that was produced using the hybridoma technology developed by Kohler and Milstein in 1975 and that specifically binds canine lymphoma cell line 17-71 (see, e.g., US 5169775A). MAb231 was shown to bind tumor cells but not normal cells and to have the murine isotype IgG2a mediating cytotoxicity in therapeutic need.

Meanwhile, a method of "caninizing (caninase)" an antibody and thus simulating a canine antibody is used. For example, Gearing et al (BMC vector Research 2013,9:226) disclose the generation of "fully caninized" anti-NGF monoclonal antibodies by converting existing murine anti-NGF monoclonal antibodies to recombinant caninized anti-NGF mAbs using an algorithm based on the expressed canine immunoglobulin sequence.

To the best of the inventors' knowledge, neither a fully synthetic canine antibody library nor any other reliable canine antibody library with a predetermined, diverse VH/VL composition has been previously disclosed. In the present invention, rational analysis of naturally occurring canine antibody sequences and complex design of libraries yielded the first fully synthetic canine antibody library that could be widely used in biomedical research. In addition, members of the library (members) were selected for beneficial properties such as high monomer content and high thermostability.

As a method for screening a synthetic canine antibody library, display on phage, escherichia coli (e.coli), yeast, or the like can be used. In preferred phage display, for example, the antibody is presented as a fusion polypeptide on the surface protein of the phage. The antibody-displaying phage particles are contacted with the target molecule of interest (e.g., immobilized on a solid phase (e.g., microtiter plate, magnetic beads, etc.) or in solution) for affinity selection. Phages expressing antibodies with affinity for the target molecule are selected, and phages with antibodies that do not bind to the target molecule are washed away in the selection cycle (selection round), which is commonly referred to as "(bio-panning)". In phage display, antibodies presented on selected phage correspond one-to-one to genes encoding the antibodies, and thus, antibodies of interest can be easily identified. Moreover, genes encoding antibodies can be easily amplified, and thus, phage display is widely used as a method for screening and isolating antibodies from large libraries.

Disclosure of Invention

Since immunoglobulin sequences encoded by canine germline sequences are not expected to be immunogenic in dogs, we sought germline immunoglobulin sequences that represent the most abundant class of canine antibodies.

According to Bao et al (vectoriary Immunology and Immunology 137(2010)64-75), the canine antibody VH gene bank (gene reptoire) comprises 80 VH segments (of which 41 are functional and 39 are pseudogenes), 6 DH and 3 JH segments. VH genes are formed by a combination of different V, D and J gene segments in the VH gene repertoire and are linked by the addition or deletion of short coding sequences at the VD and VJ junctions to increase antibody diversity. VDJ recombination that occurs prior to entry of germinal centers is not completely random, but rather specific VH genes are used more frequently than other VH genes.

In dogs, 1-VH62(Vs624) and 1-VH44(Vs635) were identified as the most frequently used VH segments in splenic B cells, with 27% 1-VH62 and 23.4% 1-VH44 (Bao et al, 2010). All remaining VH were used at a frequency of less than 11%. In dogs, there are three VH families (VH1, VH2 and VH3), most of which belong to the VH1 family, while 2-VH51, 2-VH64 and 2-VH66 belong to the VH2 family, 3-VH80 represents the only VH3 family member.

The light chain of antibodies in canines is also based on variable regions encoded by V and J gene segments and constant regions encoded by the κ and λ genes. Canine family immunoglobulin lambda sequences can be divided into four VL lambda chain families [ V-I (GenBanK accession XM845300), V-II (GenBank accession XM543519), V-III (GenBank accession XM844188) and V-IV (GenBank accession XM844237) ]. C. Family immunoglobulin kappa sequences can also be divided into four VL kappa chain families: V-I (Gen Bank accession XM849621), V-II (Gen Bank accession XM844874), V-III (Gen Bank accession XM849629) and V-IV (Gen Bank accession XM849668) sequences (Braganza et al, Veterinary Immunology and bimunography 139(2011) 27-40). Dogs were found to express 90% of lambda light chains and only 10% of kappa light chains (Braganza et al, veteriary Immunology 139(2011) 27-40). However, to date, the distribution and arrangement of the particular light chains in dogs and the VH/VL combinations present in dogs have been characterized to a very small extent.

Based on the distribution of VH genes and sequence similarity analysis described in the literature, specific VH genes were selected for the synthesis of canine antibody libraries. For the VL gene, the information provided in Braganza et al, 2011 and available inhttp:// vgenerepertoire.orgThe consensus sequence based on 86 lambda light chain germline sequences and 29 kappa light chain germline sequences obtained above was used to select a particular light chain germline sequence for synthesizing a canine antibody library.

We tested five representative canine VH germline sequences and six representative canine VL germline sequences (4 λ VL, 2 κ VL). Of the 30 possible VH/VL combinations, we identified 6 combinations that exhibited the following beneficial properties: (i) they show high display rates in the Fab format at the tip of filamentous phage; (ii) they are expressed in Fab form in soluble form with high monomer content; and (iii) is expressed in IgG form with high monomer content.

The selection of the framework is chosen to optimize the chance of obtaining the following antibodies: the antibodies possess advantageous biophysical properties and do not suffer from the disadvantages of antibodies derived from synthetic libraries that have not undergone in vivo mutations. As exemplified herein, such advantageous, desirable biophysical properties include, for example, higher stability and low aggregation propensity.

For the six most favorable VH/VL combinations, the L-CDR3 and H-CDR3 regions were replaced by highly diverse L-CDR3 and H-CDR3 library cassettes (library cassettes), respectively, to obtain greater than 5X 10⁹Overall library size of individual members. The respective restriction sites were implemented to enable L-CDR3 and H-CDR 3-library cassette insertion.

In addition, adverse post-translational modification (PTM) sites were removed from specific germline sequences to further optimize the expression and biophysical properties of the individual VH or VL genes and the corresponding proteins.

It is important to be aware of the post-translational modifications that occur in antibody preparations for therapeutic applications. PTM does not necessarily occur in antibody samples generated for initial in vitro characterization, but PTM may occur in antibody samples at high concentrations and long term storage conditions, but also in vivo. Thus, PTMs can interfere with antibody stability and/or homogeneity and may lead to loss of antibody functionality. Examples of PTMs include, but are not limited to, oxidation (Met, Trp, His), deamidation (Asn, gin), isomerization (Asp), or N-linked glycosylation (Asn).

The present disclosure provides synthetic canine antibody libraries, preferably libraries comprising at least one germline VH1 region and at least one member of a germline VL region.

In one aspect, the libraries provided herein comprise at least two germline VH1 regions and at least two members of a germline VL region.

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises members of at least one of the following germline VH1 regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.: 2).

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises members of at least two of the following germline VH1 regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.: 2). In another aspect, provided herein is a synthetic canine antibody library, wherein the library further comprises members of at least two germline VL regions.

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises at least one of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13). In one embodiment of the present disclosure, the germline VL region is selected from the group consisting of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises at least two of the following germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.:2) and comprise at least two of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.:2) and comprise the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: at least one of germline VH regions Vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.:2), and at least one of the following germline VL regions Vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: at least two of germline VH regions Vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.:2), and at least two of the following germline VL regions Vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1) and Vs635(SEQ ID No.:2), and the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs323(λ) (SEQ ID No.:16) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein are synthetic canine antibody libraries wherein post-translational modification (PTM) sites have been removed from germline VH regions or one or more of germline VL regions.

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises VH regions: vs618(SEQ ID No.:4), Vs 624-PTM-minus (SEQ ID No.:6) and Vs 635-PTM-minus (SEQ ID No.:7), and includes a VL region: vs236(κ) (SEQ ID No.:12), Vs 323-PTM-Low (λ) (SEQ ID No.:18) and Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises: VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs 323-PTM-low (λ) (SEQ ID No.:18), VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), and VH/VL combinations of VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs365(λ) (SEQ ID No.: 13).

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises members of at least two of the following germline VH1 regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.: 2). In another aspect, provided herein is a synthetic canine antibody library, wherein the library further comprises members of at least two germline VL regions.

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises at least one of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In one embodiment of the present disclosure, the germline VL region is selected from the group consisting of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In one aspect, provided herein is a synthetic canine antibody library, wherein the library comprises at least two of the following germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.:2) and comprises at least two of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.:2), and the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: at least one of the following germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.:2), and at least one of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: at least two of the following germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.:2), and at least two of the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library consists of: germline VH regions: vs618(SEQ ID No.:4), Vs624(SEQ ID No.:1), Vs628(SEQ ID No.:5) and Vs635(SEQ ID No.:2), and the following germline VL regions: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs323(λ) (SEQ ID No.:16), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein are synthetic canine antibody libraries wherein post-translational modification (PTM) sites have been removed from germline VH regions or one or more of germline VL regions.

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises VH regions: vs618(SEQ ID No.:4), Vs 624-PTM-less (SEQ ID No.:6), Vs 628-PTM-less (SEQ ID No.:10) and Vs 635-PTM-less (SEQ ID No.:7), and VL region: vs236(κ) (SEQ ID No.:12), Vs321(λ) (SEQ ID No.:14), Vs 323-PTM-minus (λ) (SEQ ID No.:18), Vs365(λ) (SEQ ID No.:13) and Vs843(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein the library comprises one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs-843 (λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 6318), VH/VL combination of VH/VL region Vs 1-PTM-less (V4) and VL combination of VH region V-VL region 321, VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365(λ) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs 3(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination comprise one or more of the VH/VL combinations disclosed herein.

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of the variable heavy chain and variable light chain combination comprise one or more of the following VH/VL combinations: VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs 323-PTM-low (λ) (SEQ ID No.:18), VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13) and VH/VL combinations of VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of one or more of the following VH/VL combinations: VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs 323-PTM-low (λ) (SEQ ID No.:18), VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13) and VH/VL combinations of VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of one or more of the VH/VL combinations disclosed herein.

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination comprise at least 2, at least 3, at least 4, at least 5, at least 6 of the following VH/VL combinations: VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs 323-PTM-low (λ) (SEQ ID No.:18), VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13) and VH/VL combinations of VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of at least 2, at least 3, at least 4, at least 5, at least 6 of the following VH/VL combinations: VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs 323-PTM-low (λ) (SEQ ID No.:18), VH/VL combinations of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combinations of VH1 region Vs 624-PTM-low (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13) and VH/VL combinations of VH1 region Vs 635-PTM-low (SEQ ID No.:7) and VL region Vs365(λ) (SEQ ID No.: 13).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of at least 2, at least 3, at least 4, at least 5, at least 6 of the VH/VL combinations disclosed herein.

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (VL No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V region No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL 618 (V) and VL combination of VH/VL region Vs-843 (V) and VL combination of VH/VL region Vs-V843 (λ) (SEQ ID No.:15), VH/VL combination of VH/VL region V1 region V-PTM-less (VL combination of SEQ ID No.:18), VH/VL combination of VH1 and VL combination of VH-VL region Vs-V, VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365(λ) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs 3(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library wherein at least 50% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination comprise one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VH/VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 1 and VL combination of VH-PTM-V region V-VL region V-No. (λ) (SEQ ID No.:18), VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236 (kappa) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16 of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (VL No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V region No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL 618 (V) and VL combination of VH/VL region Vs-843 (V) and VL combination of VH/VL region Vs-V843 (λ) (SEQ ID No.:15), VH/VL combination of VH/VL region V1 region V-PTM-less (VL combination of SEQ ID No.:18), VH/VL combination of VH1 and VL combination of VH-VL region Vs-V, VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236(κ) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365(λ) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs 3(λ) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library, wherein at least 50% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16 of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VH/VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 1 and VL combination of VH-PTM-V region V-VL region V-No. (λ) (SEQ ID No.:18), VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236 (kappa) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library wherein at least 80% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination comprise one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VH/VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 1 and VL combination of VH-PTM-V region V-VL region V-No. (λ) (SEQ ID No.:18), VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236 (kappa) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library wherein at least 50% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VH/VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 1 and VL combination of VH-PTM-V region V-VL region V-No. (λ) (SEQ ID No.:18), VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236 (kappa) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 15).

In another aspect, provided herein is a synthetic canine antibody library wherein at least 80% of the antibodies or functional fragments comprise a variable heavy chain and variable light chain combination, wherein the framework regions of said variable heavy chain and variable light chain combination consist of one or more of the following VH/VL combinations: VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs236(κ) (SEQ ID No.:12), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs365(λ) (SEQ ID No.:13), VH/VL combination of VH1 region SEQ ID No. (SEQ ID No.: 635-PTM) and VL combination of VH/VL region Vs365(λ) (SEQ ID No.:13), VH1 region Vs 635-PTM-less (V No.:7) and VL combination of VH/VL region V365 (V13) (SEQ ID No.:13), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 635-PTM-less (SEQ ID No.:7) and VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs618(SEQ ID No.:4) and VL region Vs 323-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH1 region Vs 624-PTM-less (SEQ ID No.:6) and VL combination of VH/VL region Vs-PTM-less (λ) (SEQ ID No.:18), VH/VL combination of VH/VL region 52 (V) and VL combination of VH-VL region Vs843(λ) (SEQ ID No.:15), VH/VL combination of VH1 region Vs 1 and VL combination of VH-PTM-V region V-VL region V-No. (λ) (SEQ ID No.:18), VH1 region Vs 624-PTM-few (SEQ ID No.:6) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs321(λ) (SEQ ID No.:14) VH/VL combinations, VH1 region Vs 635-PTM-few (SEQ ID No.:7) and VL region Vs236(κ) (SEQ ID No.:12) VH/VL combinations, VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs236 (kappa) (SEQ ID No.:12), VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs365 (lambda) (SEQ ID No.:13) and VH1 region Vs 628-PTM-low (SEQ ID No.:10) and VL region Vs3 (lambda) (SEQ ID No.: 15).

In one aspect, provided herein are synthetic canine antibody libraries, preferably such libraries comprising members of at least one germline VH1 region and/or at least two germline VL regions, wherein substantially all VH/VL combinations of the library are efficiently displayed in Fab form.

In one aspect, the VH/VL combination of the library is displayed in Fab format，Rate of displayIs at least 0.5 Fab/phage.

In one aspect, provided herein are synthetic canine antibody libraries, preferably such libraries that include at least one germline VH1 region and/or members of at least two germline VL regions, wherein substantially all VH/VL combinations are expressed in Fab form in e. In one embodiment, the VH/VL combination expressed in Fab form in E.coli has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% ofMonomer content。

In one aspect, provided herein are synthetic canine antibody libraries, preferably such libraries that include members of at least one germline VH1 region and/or at least two germline VL regions, wherein substantially all VH/VL combinations are expressed in the mammalian system as IgG. In one embodiment, the VH/VL combination expressed in an IgG format in a mammalian system has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% ofMonomer content。

In one aspect, provided herein are synthetic canine antibody libraries, preferably such libraries that include members of at least one germline VH1 region and/or at least two germline VL regions, wherein substantially all VH/VL combinations are thermostable.

The disclosure also provides a collection of nucleic acid molecules encoding the antibodies of the synthetic canine antibody library.

The disclosure also provides vectors encoding the nucleic acid molecules.

The disclosure also provides recombinant host cells comprising the nucleic acid molecules or vectors.

The present disclosure also provides a method of isolating an antibody specific for an antigen, the method comprising the steps of:

(a) contacting a synthetic canine antibody library of the present disclosure with an antigen;

(b) removing members of the library that do not bind to (or are not specific for) the antigen; and

(c) recovering the members of the library that bind to (or are specific for) the antigen.

The present disclosure also provides antibodies isolated from the aforementioned libraries or according to the aforementioned methods.

Drawings

FIG. 1 shows a schematic view of a

The identity tabular analysis of the 41 canine VH genes showed distance values for all possible sequence pairs of sequences included in the alignment. The distance score between pairs of sequences (i.e., 100 minus the identity score) is shown. The identity score between sequence pairs is the percentage of identical residues among all unnotched (ungapped) positions between the sequence pairs.

FIG. 2

The identity tabular analysis of 29 canine vk genes showed distance values for all possible sequence pairs of sequences included in the alignment. The distance score between pairs of sequences (i.e., 100 minus the identity score) is shown. The identity score between sequence pairs is the percentage of identical residues among all unnotched positions between the sequence pairs.

FIG. 3

The identity tabular analysis of 86 canine V λ genes showed distance values for all possible sequence pairs of sequences included in the alignment. The distance score between pairs of sequences (i.e., 100 minus the identity score) is shown. The identity score between sequence pairs is the percentage of identical residues among all unnotched positions between the sequence pairs.

FIG. 4

Western blot analysis of the VH/VL construct subset (subset) to assess Fab-display rates.

FIG. 5

Densitometric (densitometric) analysis of the blots shown in figure 4, showing appropriate display rates (typically 0.5-2 Fab/phage) for efficient phage display.

FIG. 6

The relative Fab expression of the 30 VH/VL combinations in bacterial cell lysates was analyzed by ELISA, where the expression level of each VH/VL pair was determined relative to the expression of a reference Fab control. Essentially all of the Fab VH/VL pairs tested showed a relative expression of at least 0.5 of the control. Lambda clones had on average the highest relative Fab expression level. Sample numbers are compiled in table 3.

FIG. 7

Fab expression yields (shown as bar graphs; left axis) and monomer content (shown as dot graphs; right axis) for the 30 VH/VL combinations. Sample numbers are compiled in table 3.

FIG. 8

Study-grade IgG expression yields (shown as bar graphs; left axis) and monomer content (shown as dot graphs; right axis) for the 30 VH/VL combinations. Sample numbers are compiled in table 3.

FIG. 9

The basic part of the phage display vector pCaDis is shown to include the relevant unique restriction sites.

FIG. 10 shows a schematic view of a

The basic portion of the bacterial Fab expression vector pCaBx, including the associated unique restriction sites, is shown.

FIG. 11

High quality and correctness of canine antibody libraries as shown by the distribution of VH major genes predicted by each library composition.

FIG. 12

For the exemplary light chain CDRs, the designed amino acid profile (left column) was compared to the amino acids obtained at the corresponding positions in the library (right column), confirming the exact, correct composition of the synthetic library.

FIG. 13

After quality control of MiSeq NGS of about 790 ten thousand sequences, the designed CDR-H3 length distribution (white bars) was compared to the resulting CDR-H3 length distribution (dark bars).

FIG. 14

Dot blot visualization of Fab screening results. The signal above background for specific binding to eGFP is plotted on the x-axis (direct ELISA) and the results for Fab expression are shown on the y-axis (signal above background in Fab-capture ELISA). The shapes of the dots show the respective pan subcodes (panning subcodes).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the context of values and ranges, the term "about" or "approximately" means approximately or near the value or range so that the invention can be performed as intended, e.g., with the number or percentage of sequence homology desired, as will be apparent to those skilled in the art from the teachings contained herein. This is due at least in part to variations in culture conditions and variability in biological systems. Thus, these terms include values beyond those generated by systematic errors. These terms are used to make explicit the implicit content.

All ranges set forth herein in the summary and description of the invention are intended to include all numbers and values thereabout or between the numbers of the range. The scope of the invention is expressly intended to include all integer, decimal, and fractional values within the scope. The term "about" may be used to describe a range.

As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of IgG, IgA or IgD antibodies comprises three domains CH1, CH2 and CH3, while the heavy chains of IgM and IgE antibodies consist of four domains CH1, CH2, CH3, CH 4. Each light chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes a domain CL. The VH and VL regions may be further subdivided into hypervariable regions, called Complementarity Determining Regions (CDRs), interspersed with more conserved regions called Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The framework regions and the extent of the CDRs have been precisely determined (see Kabat,1991, J.Immunol.,147,915, 920; Chothia & Lesk,1987, J.mol.biol.196:901, 917; Chothia et Al, 1989, Nature 342:877, 883; Al-Lazikani et Al, 1997, J.mol.biol.273:927, 948). The framework regions of an antibody, i.e., the combined framework regions that make up the light and heavy chains, are used to locate and align the CDRs, which are primarily responsible for binding to the antigen.

The term "antigen-binding portion" or "fragment" of an antibody is used equally in this application. These terms refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen binding function of an antibody may be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; f (ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment consisting of the VH and CH1 domains; (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (dAb) fragments (Ward et al, 1989Nature 341:544-546) consisting of a VH domain; and an isolated Complementarity Determining Region (CDR). Preferred antigen binding portions or fragments of antibodies are Fab fragments.

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by an artificial peptide linker using recombinant methods, enabling them to be prepared as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al, 1988Science 242: 423-58426; and Huston et al, 1988Proc. Natl. Acad. Sci.85: 5879-5883). Such single chain antibodies include one or more "antigen-binding portions" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for use in the same manner as intact antibodies. Antigen-binding moieties may also be incorporated into single domain antibodies, macroantibodies (maxibodies), minibodies (minibodies), intrabodies (intrabodies), diabodies (diabodies), triabodies (triabodies), tetrabodies (tetrabodies), v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson,2005, Nature Biotechnology,23,9, 1126-1136). The antigen-binding portion of the antibody may be grafted into a polypeptide-based scaffold such as fibronectin type III (Fn3) (see U.S. patent No. 6,703,199, which describes fibronectin polypeptide mono-antibodies). The antigen-binding portion may be incorporated into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with a complementary light chain polypeptide form a pair of antigen-binding regions (Zapata et al, 1995Protein Eng.8(10): 1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term "canine antibody" refers to an antibody having variable regions in which both the framework and CDR regions are derived from canine-derived sequences. For example, both the framework and CDR regions can be derived from canine derived sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from such canine-derived sequences, e.g., canine germline sequences or mutated forms of canine germline sequences. The canine antibodies of the present invention may include amino acid residues that are not encoded by canine sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).

The term "originalCanine immune repertoire (supercare) "refers to a repertoire of nucleic acids isolated from B cells from the canine immune system that have not undergone antigen, wherein the nucleic acids encoding the antibody or functional fragment thereof have not undergone somatic hypermutation and are therefore considered to comprise nucleic acids of germline genes with concomitant v (d) J gene segment rearrangement. The repertoire (repotoreie) may be a repertoire of individuals or groups. Preferably, the immune repertoire is obtained from multiple individuals to avoid sample bias.

The term "canine immune repertoire" refers to a repertoire of nucleic acids isolated from B cells from the canine immune system. The repertoire can be individual or population and can be derived from naive B cells and/or antigen-experienced B cells. Preferably, the immune repertoire is obtained from multiple individuals to avoid sample bias.

The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen binding specificities. However, an isolated antibody that specifically binds an antigen may have cross-reactivity with other antigens. Moreover, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "isotype" refers to the class of antibodies provided by the heavy chain constant region genes (e.g., IgM, IgE, IgA, IgG, such as IgG1 or IgG 4). In the canine species, there are four IgG subclasses: IgG-A, IgG-B, IgG-C and IgG-D (L.M. Bergeron et al, Veterinyl Immunology and Immunology 157(2014) 31-41). Isoforms also include modified forms of one of these classes in which the modification is made to alter Fc function, e.g., to increase or decrease effector function or binding to Fc receptors.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "library" is meant to typically comprise more than 10³Greater than 10⁴Greater than 10⁵Greater than 10⁶Greater than 10⁷Greater than 10⁸Greater than 10⁹Or even greater than 10¹⁰A collection of different molecules of individual members. A library in the context of the present invention is a mixture of heterologous polypeptides or nucleic acids. The library is composed of members, each member having a single polypeptide or nucleic acid sequence. Sequence differences between library members are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of an organism or cell, such as a bacterium, virus, phage, animal or plant cell, transformed with a library of nucleic acids. Preferably, each individual organism or cell contains only one or a limited number of library members. Advantageously, the nucleic acid is introduced into an expression vector so as to allow expression of the polypeptide encoded by the nucleic acid. In certain aspects, the library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing an individual member of the library in nucleic acid form that can be expressed to produce its corresponding polypeptide member. Thus, a population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants (large repotoreie). The term "collection" is essentially interchangeable with the term "library" such thatThe application is as follows.

Antibody libraries can be derived from immunoglobulins or fragments thereof that are biased towards certain specificities present in immunized animals or naturally immunized or infected humans. Alternatively, the antibody library may be derived from naive immunoglobulins or fragments thereof, i.e., immunoglobulins that are not biased towards the specificity found in the immune system. Such libraries are referred to as "unbiased" libraries. In preferred embodiments, the present disclosure provides unbiased antibody libraries, i.e., libraries that are not pre-exposed to the antigen of interest. Such libraries include antibodies that bind to any potential target antigen of interest, in the absence of any bias.

Typically, immune antibody libraries are constructed from libraries of VH and VL genes cloned from source B cells by PCR (or related) based cloning techniques. In the same way, unbiased primary antibody libraries can also be generated. However, unbiased naive antibody libraries can also be generated synthetically, where the entire library is constructed entirely in vitro. Using recombinant DNA technology, it can be used to mimic the natural bias and abundance of natural antibody libraries. Such antibody libraries are referred to as "synthetic" antibody libraries. The term "fully synthetic" library refers to a library that is constructed completely, i.e., completely de novo, by DNA synthesis (e.g., by whole gene synthesis, PCR-based methods, or related DNA techniques). In such libraries, de novo constructionThe wholeDNA, i.e., the portion encoding the CDRs and the portion (e.g., framework region) encoding the portion surrounding the CDRs of the library antibody. The terms "synthesis" and "total synthesis" thus refer to a de novo source of DNA. In contrast, in a "semi-synthetic" antibody library, only part of the library antibodies are constructed de novo, while other parts, such as certain CDR regions, are derived from natural sources (for which there is a large review, see, e.g., Sidhu et al; Nat ChemBiol (2006),2,682-8). In certain aspects, the disclosure provides synthetic canine antibody libraries. In a preferred aspect, the present disclosure provides a fully synthetic canine antibody library.

The term "complete germline" refers to the nucleotide sequence of antibody genes and gene fragments as they are passed from parent to offspring by germ cells. Germline sequences differ from nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during B cell maturation. The term "germline" refers to the complete germline sequence and additionally to germline sequences that have been modified or engineered with minor mutations in the amino acid sequences, for example, for the purposes of: removal of undesired post-translational modification (PTM) sites, removal of undesired cysteines, optimization of antibodies (e.g. affinity, half-life) or introduction of desired restriction sites, or modifications caused by errors in synthesis, amplification or cloning.

The term "post-translational modification" or "PTM" refers to a modification of a protein that is typically enzymatic during or after the biosynthesis of the protein. Post-translational modifications may occur in the amino acid side chain, or at the C-or N-terminus of the protein. They can extend the chemical repertoire of 20 standard amino acids by introducing new functional groups such as phosphate, acetate, amide or methyl. Many eukaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation, which can facilitate folding of the protein, improve stability, and serve a regulatory function. Modifications occur at so-called post-translational modification sites (i.e., defined amino acid motifs), which specifically include N-linked glycosylation sites (NxS or NxT) or chemical modifications such as Asp cleavage (usually at DP), Asp isomerization (DS, DG), deamidation (NS, NG). Methionine may be oxidized upon exposure to a solvent. Modification can occur in vivo (in serum) or upon storage in formulation buffer and result in loss of antibody binding.

The term "germline variable region" refers to:

a) a nucleic acid sequence or amino acid sequence of a variable region of an antibody or functional fragment thereof encoded by a germline gene;

b) a nucleic acid sequence or amino acid sequence of a variable region of an antibody or functional fragment thereof encoded by a germline gene, wherein the nucleic acid sequence is modified by, for example, codon optimization, addition of desired restriction sites, optimized GC content, removal of undesired post-translational modification (PTM) sites, removal of undesired mRNA splice sites or removal of mRNA instability motifs, or

c) The nucleic acid sequence or amino acid sequence of the variable region of an antibody or functional fragment thereof encoded by a germline gene, but with minor mutations in the amino acid sequence, for example, for the following purposes: removal of undesired post-translational modification (PTM) sites, removal of undesired cysteines or introduction of desired restriction sites, or modifications resulting from errors in synthesis, amplification or cloning.

In the sense of the present disclosure, a "germline variable region" is a "germline VH region" or a "germline VL region". Examples of canine "germline variable regions" are shown in table 1.

The term "combination of a variable heavy chain and a variable light chain" or "VH/VL combination" refers to the combination (pairing) of one variable heavy chain and one variable light chain. Antibodies and functional fragments such as Fab include at least one variable heavy chain combined with a variable light chain, which forms an antigen binding region.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages (which are synthetic, natural and non-natural) that have similar binding properties and are metabolized in a manner similar to the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues, as described in detail below (Batzer et al, Nucleic Acid Res.19:5081,1991; Ohtsuka et al, J.biol.chem.260: 2605-.

As used herein, the term "optimized" refers to a nucleotide sequence that has been altered to encode an amino acid sequence using codons that are preferred in a production cell or organism, typically a eukaryotic cell, e.g., a pichia cell, a Chinese Hamster Ovary (CHO) cell, or a human cell. The optimized nucleotide sequence is engineered to retain, completely or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence (also referred to as the "parent" sequence). The sequences optimized herein have been engineered to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. Optimized nucleotide sequence the amino acid sequence expressed is also referred to as optimized.

The term "amino acid" refers to both natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the natural amino acids, natural amino acids being those encoded by the genetic code, and those amino acids that are later modified, e.g., hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acid, as well as to natural amino acid polymers and unnatural amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly includes conservatively modified variants thereof.

The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more identical sequences or subsequences. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over the designated region, or over the entire sequence when not specified), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or additional optional parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

The term "recombinant antibody" as used herein includes all antibodies produced, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals transgenic or transchromosomal for human immunoglobulin genes (e.g., mice) or hybridomas produced therefrom, antibodies isolated from host cells transformed to express rodent, human or canine antibodies, such as from transfectomas, antibodies isolated from recombinant, combinatorial canine antibody libraries, and antibodies produced, expressed, produced or isolated by any other means involving splicing of all or a portion of the canine immunoglobulin genes, sequences to other DNA sequences. These recombinant canine antibodies have variable regions in which the framework and CDR regions are derived from canine germline immunoglobulin sequences. However, in certain embodiments, these recombinant canine antibodies can be subjected to in vitro mutagenesis (or, when transgenic animals are used with canine Ig sequences, in vivo somatic mutagenesis) and, therefore, the amino acid sequences of the VH and VL regions of the recombinant antibodies are the following sequences: it, although derived from and related to canine germline VH and VL sequences, may not naturally occur in vivo in canine germline antibody repertoires.

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It is understood that these terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication into a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors for recombinant DNA techniques are usually in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

"display vectors" include DNA sequences that have the ability to direct the replication and maintenance of a recombinant DNA molecule extrachromosomally in a host cell (e.g., a bacterial host cell) transformed therewith. These DNA sequences are well known in the art. The display vector may be, for example, a phage vector or a phagemid vector derived from the fd, M13 or fl filamentous phage class. These vectors are capable of facilitating the display of proteins, including, for example, binding proteins or fragments thereof, on the surface of filamentous phages. Display vectors suitable for display on phage, ribosomes, DNA, bacterial cells or eukaryotic cells such as yeast or mammalian cells are also known in the art, e.g., viral vectors or vectors encoding chimeric proteins.

A "unique" restriction site is one that is present or occurs only once on a given nucleic acid molecule. Typically, such nucleic acid molecules are vectors encoding members of the libraries of the present invention.

The term "position-dependent amino acid usage" refers to the likelihood that a particular amino acid sequence will occur at a given position in a polypeptide. In the present invention, for rearranged amino acid sequences classified by individual germline genes, position-dependent amino acid usage is determined. This enables individual, precise design of CDRs within their natural germline context.

As used herein, the term "substantially all" means that the component to which it refers is more or less pure. Only small amounts or other different components are present which do not limit or influence the advantageous properties of the components. Depending on the nature of the component, substantially all may refer to at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the component.

As used herein, the term "PTM-low" refers to antibody germline VH and/or VL amino acid sequences that have been modified within Kabat H-CDR1 and/or H-CDR2 to remove potential post-translational modification (PTM) sites. Preferably, the potential PTM motifs in framework regions FR1, FR2, FR3 and FR4 are not modified.

The J region amino acid sequences of FR4 of the heavy chain, kappa light chain and lambda light chain were WGQGTLVTVSS (SEQ ID No.:37), FGAGTKVELK (SEQ ID No.:38) and FGGGTQLTVL (SEQ ID No.:39), respectively, as shown in Table 2.