Cat antibody library

Abstract

The present invention relates to and provides polypeptide libraries, such as synthetic antibody libraries, that are suitable for the selection of complete feline antibodies. The present invention also relates to synthetic nucleic acid sequences that code for individual feline antibodies or collections of feline antibodies, i.e., nucleic acid sequences that code for feline antibody libraries. Methods for producing and using such libraries are provided. In particular, the present invention relates to the preparation of libraries of complete feline antibodies by the use of pre-evaluated synthetic sequences to generate libraries of complete feline antibodies with favorable biophysical properties.

Description

[Technical field]

[0001] The present invention relates to and provides polypeptide libraries, such as synthetic antibody libraries, that are suitable for the selection of complete feline antibodies. The present invention also relates to synthetic nucleic acid sequences that code for individual feline antibodies or collections of feline antibodies, i.e., nucleic acid sequences that code for feline antibody libraries. Methods for producing and using such libraries are provided. In particular, the present invention relates to the preparation of libraries of complete feline antibodies by the use of pre-evaluated synthetic sequences to generate libraries of complete feline antibodies with favorable biophysical properties. [Background technology]

[0002] In the following discussion, certain articles and methods are described for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right, under applicable statutory provisions, to demonstrate, where appropriate, that the articles and methods referred to herein do not constitute prior art.

[0003] It has been more than 30 years since the first monoclonal antibody (mAb), Orthoclone OKT3, was approved by the United States Food and Drug Administration (US FDA) in 1986 to prevent kidney transplant rejection (Ecker et al. 2015). Since then, the technology for generating and optimizing human therapeutic mAbs has improved significantly, making them a promising class of new drugs developed in recent years. The market for mAbs has experienced substantial growth, and drugs are available to treat a variety of human diseases, including many cancers, autoimmune, metabolic, and infectious diseases. As of December 2021, over 100 therapeutic mAbs have been approved by regulatory agencies, and over 80 are in late-stage clinical development (Kaplon and Reichert 2021).

[0004] Companion animals, such as dogs and cats, develop diseases that are similar or even the same as human underlying pathological mechanisms. Surprisingly, however, the use of therapeutic mAbs in animal health has only recently begun to develop, with only a few approved antibodies available to date (e.g., https: / / www.zoetisus.com / products / dogs / cytopoint / #). The limited progress reflects the fact that the generation of species-specific mAbs is technically challenging, and experience in developing antibodies for companion animals is only slowly developing.

[0005] Only a few technical approaches are available for generating therapeutic antibodies for companion animals, e.g. dogs or cats, namely modification of existing compounds and use of transgenic animals. Methods have been disclosed for "caninizing" or "felinizing" antibodies. For example, the generation of a caninized anti-NGF mAb from an existing rat antibody has been described, using an algorithm for comparison of the starting antibody with the canine immunoglobulin sequence to identify the modifications necessary to make the sequence pet-like (Gearing et al. 2013). A similar method was also applied for the generation of feline antibodies (Gearing et al. 2016). However, even subtle changes in the protein sequence of the antibody can result in significant loss of efficacy and altered biophysical properties, making such methods time-consuming and prone to failure. More advanced techniques concern transgenic rodents expressing canine immunoglobulins (Wabl 5 / 23 / 2017). The disadvantages are the need to sacrifice animals for the initial antibody discovery process and the fact that the immunization process is poorly controllable.

[0006] In this respect, in vitro selection methods, such as phage display, offer great advantages, since they allow for a tailored antibody selection process. Very recently, a synthetic phage display library containing complete canine antibody fragments has been disclosed (Tiller et al. 6 / 21 / 2018). This library was constructed in preselected and tested heavy and light chain pairings based on germline genes, and is therefore expected to result in non-immunogenic antibodies when administered to canine patients. Diversity was introduced in the CDR3 of both the heavy and light chains, and the CDRs were designed according to the natural amino acid and length distribution of the analyzed canine antibody sequences. Members of the library were also selected for advantageous properties, such as high monomer content and high thermal stability.

[0007] Over recent years, there has been growing interest in cats not only as companion animals, but also as models for specific diseases. To the inventors' knowledge, a fully synthetic feline antibody library with predefined heavy and light chain composition, as well as any other authentic collection of feline antibodies, has not yet been disclosed.

[0008] Within the present invention, the rational analysis of experimentally selected naturally occurring feline antibody sequences and the elegant design of a library have resulted in the first fully synthetic feline antibody library that can be used for biomedical research.

[0009] The feline immune system achieves diversity within the antibody repertoire by V(D)J recombination, a process in which a series of variable (V), diversity (D), and joining (J) genes recombine to create the variable domains of antibodies. Additional processes, such as somatic hypermutation, insertion, and deletion, further increase the diversity of antibody variable regions. Cats also exhibit a classical mammalian organization of the IGH genome, with the IG locus described (Cho et al. 1998), which consists of 24 IGHV genes, 13 IGKV genes, and 47 IGLV genes (Olivieri et al. 2014). Domestic cats have been studied immunologically in detail (Baldwin and Denham 1994, Kanai et al. 2000, Klotz et al. 1985, Schultz et al. 1974). Two allelic sequences, designated feline IgG1a and 1b, have been described (Kanai et al. 2000), which are expected to function similarly to human IgG1 and induce strong effector functions in vivo (Strietzel et al. 2014). The same authors report the existence of a rare IgG sequence, now designated feline IgG2. This additional IgG does not bind recombinant fFcγRI or fFcγRIII and has negligible binding to C1q, indicating a lack of effector function. Also disclosed are the constant regions of feline kappa (AF198257.1) and lambda (XM 003994910.1, E07339.1).

[0010] However, the actual expressed feline antibody repertoire is less well described. Next generation sequencing (NGS) data of feline antibodies has revealed some insights into the architecture and abundance of feline antibodies, as well as into CDR3 diversity (Steiniger et al. 2017). Even more strikingly than in dogs (Steiniger et al. 2014), the feline antibody repertoire is dominated by one heavy chain subgroup that is homologous to human VH3. Approximately 99% of all analyzed sequences in this study were clustered in this subgroup. A characteristic feature described for many domesticated species, including cats, dogs, sheep, cattle, and horses, is the preferential expression of lambda light chains (Sitnikova and Su 1998, Steiniger et al. 2017). These results were also confirmed by an in-house performed NGS analysis, which highlighted a significant bias towards the use of VH3 type heavy chains and lambda light chains.

[0011] The HCDR3 length distribution of feline antibodies shows an approximately normal distribution (Steiniger et al. 2017) and is similar in length to the canine repertoire (Steiniger et al. 2014).

[0012] Very few feline therapeutic antibodies have been described (Doki et al. 2016, Gruen et al. 2016, Enomoto et al. 2019, Walters et al. 2021). Of note, the candidates described are derived from rodent antibodies that have been subjected to modifications to make them compatible with the feline immune system. Due to their origin, the light chains are of the kappa subtype, which is uncommon in cats.

[0013] In conclusion, the natural pairing of feline antibody heavy and light chains is poorly characterized.

[0014] However, the identification of stable VH / VL combinations is of utmost importance, as these represent the basis for a functional antibody library. Naturally occurring immunoglobulin sequences encoded by cats are not expected to be immunogenic in cats. Summary of the Invention

[0015] Heavy and light sequences were PCR amplified from feline bone marrow cDNA. The resulting sequences were cloned into a phage display vector to generate phages containing random pairing of VH and VL sequences in a single chain format (scFv) and containing a FLAG tag. Following phage preparation, an anti-FLAG pulldown was performed to enrich for only phage particles displaying intact FLAG-scFv fragments. Phage products were cloned into a bacterial expression vector and the expression levels of individual feline scFvs were determined by ELISA. Clones with high expression levels were then sequenced and converted to Fab format. Expression of soluble Fab fragments was confirmed by ELISA. Following these selection steps, feline antibody sequences were identified that met the following criteria: (i) were compatible with phage display, (ii) showed good expression levels as scFvs, and (iii) showed good expression levels as Fab fragments, and therefore represented ideal scaffolds for constructing synthetic feline antibody libraries with the preselected and tested framework regions. Selection of VH and VL sequences and their respective pairing based on the described approach, including soluble scFv expression in a first step and Fab fragments in a subsequent step, resulted in a library with surprisingly good conversion rates from clones initially identified in scFv format to expression as Fab and as full-length IgG antibodies. To the inventors' knowledge, this is the first experimental approach to identify a collection of stable feline heavy and light chain combinations and develop an in vitro complete feline antibody selection technology.

[0016] Thus, based on the observed properties and determined sequences, in one aspect the invention provides a polypeptide library comprising VL polypeptide members comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, The present invention provides a polypeptide library comprising at least one sequence selected from sequence number 108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, and / or SEQ ID NO:137. Preferably, at least one sequence comprised within a VL FW1-CDR1-FW2-CDR2-FW3 segment comprised within a VL polypeptide is selected from SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, and / or SEQ ID NO:135.

[0017] In a further aspect, the invention provides a polypeptide library comprising a VH polypeptide member comprising a FW1-CDR1-FW2-CDR2-FW3 segment, wherein the FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO: No. 166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:202. Preferably, at least one sequence comprised within a VH FW1-CDR1-FW2-CDR2-FW3 segment comprised within a VH polypeptide is selected from: SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150 SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, and SEQ ID NO:200.

[0018] The preferred VL FW1-CDR1-FW2-CDR2-FW3 and VH FW1-CDR1-FW2-CDR2-FW3 segments showed high expression levels as scFv and Fab fragments.

[0019] More specifically, the present invention relates to a polypeptide library comprising at least one VL / VH polypeptide member combination, wherein the at least one VL / VH polypeptide member combination is selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:77 and SEQ ID NO:142, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:79 and SEQ ID NO:144, SEQ ID NO:80 and SEQ ID NO:145, SEQ ID NO:81 and SEQ ID NO:146, SEQ ID NO:82 and SEQ ID NO:147, and SEQ ID NO:149. 7, SEQ ID NO:83 and SEQ ID NO:148, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:87 and SEQ ID NO:152, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:89 and SEQ ID NO:154, SEQ ID NO:90 and SEQ ID NO:155, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:93 and SEQ ID NO:158, SEQ ID NO:94 and SEQ ID NO:159, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:96 and SEQ ID NO:161, SEQ ID NO:97 and SEQ ID NO:162, SEQ ID NO:98 and SEQ ID NO:163, SEQ ID NO:99 and SEQ ID NO:164, SEQ ID NO:100 and SEQ ID NO:165, SEQ ID NO:101 and SEQ ID NO:166, SEQ ID NO:102 and SEQ ID NO:167, SEQ ID NO:103 and SEQ ID NO:168, SEQ ID NO:104 and SEQ ID NO:169, SEQ ID NO:105 and SEQ ID NO:170, SEQ ID NO:106 and SEQ ID NO:171, SEQ ID NO:107 and SEQ ID NO:172, SEQ ID NO:108 and SEQ ID NO:173, SEQ ID NO:109 and SEQ ID NO:174, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:111 and SEQ ID NO:176, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:1 13 and SEQ ID NO:178, SEQ ID NO:114 and SEQ ID NO:179, SEQ ID NO:115 and SEQ ID NO:180, SEQ ID NO:116 and SEQ ID NO:181, SEQ ID NO:117 and SEQ ID NO:182, SEQ ID NO:118 and SEQ ID NO:183, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:120 and SEQ ID NO:185, SEQ ID NO:121 and SEQ ID NO:186, SEQ ID NO:122 and SEQ ID NO:187, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:124 and SEQ ID NO:189, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:126 and SEQ ID NO:191, SEQ ID NO:127 and SEQ ID NO:192,A polypeptide library is provided comprising a sequence combination selected from SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:130 and SEQ ID NO:195, SEQ ID NO:131 and SEQ ID NO:196, SEQ ID NO:132 and SEQ ID NO:197, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:136 and SEQ ID NO:201, and / or SEQ ID NO:137 and SEQ ID NO:202. Preferably, the polypeptide library comprises at least one VL / VH polypeptide member combination, wherein at least one VL / VH polypeptide member combination is selected from SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:91 and SEQ ID NO:154. and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, and / or SEQ ID NO:135 and SEQ ID NO:200.

[0020] As a further aspect of the invention, based on the alignment of the preferred members identified above (Figure 1), the inventors have determined a consensus sequence for a preferred polypeptide VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4, comprising: - FW1 is X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, - HCDR1 is X30, X31, X32, X33, X34, X35, - FW2 is X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, - HCDR2 is X49, X50, X51, X52, X52a, X53, X54, X55, X56, X57, X58, X59, X60, - FW3 is X61, X62, X63, X64, X65, X66, X67, X68, X69, X70, X71, X72, X73, X74, X75, X76, X77, X78, X79, X80, X81, X82, X83, X84, X85, X86, X87, X88, X89, X90, X91, X92, X93, X94, X95, X96, X97, - HCDR3 is X98, X99, X100, X101, X101a, X101b, X101c, X101d, X101e, X101f, X101g, X102, X103, X104, - FW4 is X105, X106, X107, X108, X109, X111, X112, X113, X114, X115, A consensus sequence could be derived where X1 denotes the first amino acid position, X2 denotes the second amino acid position, and so on, and the nature of the amino acids at corresponding positions are defined as follows: [Table A-1] [Table A-2] (Amino acids in bold and underlined are preferred amino acids, and "-" defines an empty position)

[0021] Thus, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence: (D / Q / Y / E)(V / E)(Q / L / R)LV(E / Q)SGGD(L / R)V(K / Q)PGGSLRL(T / I / A)C(V / M)(A / G)SGF(T / P / I / N)(F / V / L) XaaXaaXaaXaaXaaXaaWVRQ(A / T)PG(K / M)GLQWVXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa I / V)S(R / K)D(N / D)(A / P / V)(K / R / M / E)NTL(Y / L)LQM(N / T / D)(S / G / N)LKTED(T / A / M)ATYYC(A / S / T)(R / K / N / T / G)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWG(Q / R)G(A / T / V)(L / P)VTVSS; The amino acids of the HCDRs are designated as variable amino acids (Xaa), which can be any one of A (Ala), R (Arg), N (Asn), D (Asp), C (Cys), Q (Gln), E (Glu), G (Gly), H (His), I (Ile), L (Leu), K (Lys), M (Met), F (Phe), P (Pro), S (Ser), T (Thr), W (Trp), Y (Tyr), or V (Val).

[0022] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence: (D / Q / Y / E)(V / E)(Q / L / R)LV(E / Q)SGGD(L / R)V(K / Q)PGGSLRL(T / I / A)C(V / M)(A / G)SGF(T / P / I / N)(F / V / L)(S / R / G / T / N)(S / R / G / N / D / L)(Y / H / F / D)(G / W / D / E / A / S / Y)M(S / N / T / D / H)WVRQ(A / T)PG(K / M)GLQWV(A / S / T)(A / S / Y / G / T / Q / D)(I / N / L)(S / G / R / D / A)(G / F / Y / S / N / D / R)(S / T / D / G / N)G(G / S / I / H / D / A)(S / A / T / V / N)(T / I)(Y / A / G / N / H)Y(A / S / G / I)DSVKGRFT(I / V)S(R / K)D(N / D)(A / P / V)(K / R / M / E)NTL(Y / L)LQM(N / T / D)(S / G / N)LKTED(T / A / M)ATYYC(A / S / T)(R / K / N / T / G)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWG(Q / R)G(A / T / V)(L / P)VTVSS (SEQ ID NO: 233), may have, The amino acids of HCDR 1 and 2 are defined in the sequence.

[0023] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence, DVQLVESGGDLVKPGGSLRLTCVASGFTFXaaXaaXaaXaaXaaXaaWVRQAPGKGLQWVXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaDSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCARXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWGQGALVTVSS (SEQ ID NO: 234), or DVQLVESGGDLVKPGGSLRLTCVASGFTFSSYGMSWVRQAPGKGLQWVAAISGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCARXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWGQGALVTVSS (sequence number 235).

[0024] Also for the VL polypeptide, as a further aspect of the invention, we have determined, based on the alignment of the preferred VL members identified above (Figure 2), a consensus sequence for the preferred polypeptide VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4, - FW1 is Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, - LCDR1 is Y23, Y24, Y24a, Y24b, Y24c, Y24d, Y24e, Y24f, Y25, Y26, Y27, Y28, Y29, FW2 is Y30, Y31, Y32, Y33, Y34, Y35Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y44a, Y44b, Y44c, Y44d, - LCDR2 is Y45, Y46, Y47, Y48 Y49, Y50, Y51, Y52, Y53, Y54, FW3 is Y55, Y56, Y57, Y58, Y59, Y60, Y61, Y61a, Y61b, Y62, Y63, Y64, Y65, Y66, Y67, Y68, Y69, Y70, Y71, Y72, Y73, Y74, Y75, Y76, Y77, Y78, Y79, Y80, Y81, Y82, Y83; - HCDR3 is Y84, Y85, Y86, Y87, Y88, Y89, Y90, Y90a, Y90b, Y90c, Y91, Y92, - FW4 is Y93, Y94, Y95, Y96, Y97, Y98, Y99, Y100, Y101, Y102, A consensus sequence could be derived in which Y1 denotes the first amino acid position, Y2 denotes the second amino acid position, and so on, and the nature of the amino acids at corresponding positions are defined as follows: [Table B-1] [Table B-2] (Amino acids in bold and underlined are preferred amino acids, and "-" defines an empty position)

[0025] Thus, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following sequence: Q(S / P / E)(V / G)(P / L / V)(T / N)Q(P / E)(P / S / T)S(V / L / M)S(G / A / T)(A / G / S / T)( L / P)G(Q / G / T)(R / A / T / K)(V / I)(T / R)(I / M / L)(S / T)CXaaXaaXaaXaaXaaXaaXaa XaaXaaXaaXaaXaaXaaXaaW(Y / V)(Q / R)Q(I / V / L / K / H)(P / S)(G / R / Y)(M / K / R / Q / T / S)(A / P / R)(P / F)(K / Q / S)(T / L / A / V / Y)(L / I / V / F)(I / V / L)(Y / V / G / H)(- / Y)( - / Y)(- / S)(- / D)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(D / A / N / S)R(F / I / L / V)S GS(K / L)(- / D)(- / A)(S / V)(G / A)(S / N / T / Q / I / A)(T / K / A)(G / A)(S / T / L / V)L(T / A / L)(I / V)(T / S)G(L / P / A)Q(A / S / T / P)EDE(A / G / V)(D / N)Y(Y / H)CXaaXaaXaaXa aXaaXaaXaaXaaXaaXaaXaaXaa(F / I)G(G / R)GTH(L / V)(T / S)VL (SEQ ID NO: 236); The amino acids of LCDR are indicated as variable amino acids (Xaa), which can be any one of A (Ala), R (Arg), N (Asn), D (Asp), C (Cys), Q (Gln), E (Glu), G (Gly), H (His), I (Ile), L (Leu), K (Lys), M (Met), F (Phe), P (Pro), S (Ser), T (Thr), W (Trp), Y (Tyr), or V (Val).

[0026] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following sequence: Q(S / P / E)(V / G)(P / L / V)(T / N)Q(P / E)(P / S / T)S(V / L / M)S(G / A / T)(A / G / S / T)(L / P)G(Q / G / T)(R / A / T / K) (V / I)(T / R)(I / M / L)(S / T)C(T / A / S / G)(G / S / L)(S / T / -)(S / A / G / T / R / -)(S / N / G / P / E / -)(N / A / F / V / -)(I / N / V / S / -)(G / T / V / -)(V / R / I / S / T / G / A / -)(G / V / A / D / S / T)(N / D / G / T / S / M / V / K / Y)(Y / S / G / -)(V / A / I)(S / N / G / D / Y)W(Y / V)(Q / R)Q(I / V / L / K / H)(P / S)(G / R / Y)(M / K / R / Q / T / S)(A / P / R)(P / F)(K / Q / S)(T / L / A / V / Y) (L / I / V / F)(I / V / L)(Y / V / G / H)(- / Y)(- / Y)(- / S)(- / D)(G / A / S / D / E / R / Y)(N / S / D / T)(S / N / T / D / R / K / F)( N / R / K / D / E / Y / H)(R / Q / L)(P / N / G)(S / P)(G / S / E)VP(D / A / N / S)R(F / I / L / V)SGS(K / L)(- / D)(- / A)(S / V)(G / A)(S / N / T / Q / I / A)(T / K / A)(G / A)(S / T / L / V)L(T / A / L)(I / V)(T / S)G(L / P / A)Q(A / S / T / P)EDE(A / G / V)(D / N)Y(Y / H)CXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(F / I)G(G / R)GTH(L / V)(T / S)VL (SEQ ID NO: 237); The amino acids of LCDR 1 and 2 are defined in the sequence.

[0027] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following sequence: QSVPTQPPSVSGALGQRVTISCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa aXaaXaaXaaXaaDRFSGSKSGSTGSLTITGLQAEDEADYYCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaFGGGTHLTVL (SEQ ID NO: 238) or It may have the structure: QSVPTQPPSVSGALGQRVTISCTGSSSNIGVGNYVSWYQQIPGMAPKTLIYGNSNRPSGVPDRFSGSSKSGSTGSLTITGLQAEDEADYYCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaFGGGTHLTVL (sequence number 239).

[0028] For purposes of integration, introduction of restriction sites, removal of PTM sites, etc., the originally specified VH FW1-CDR1-FW2-CDR2-FW3 and VL FW1-CDR1-FW2-CDR2-FW3 segments were modified as shown in Figures 9 and 10.

[0029] Accordingly, in a further aspect, the present invention relates to a polypeptide library comprising VL polypeptide members comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment comprises at least one sequence selected from SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224, and / or SEQ ID NO:224; and / or to a polypeptide library comprising VH polypeptide members comprising a VH FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VH FW1-CDR1-FW2-CDR2-FW3 segment comprises at least one sequence selected from SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231.

[0030] In a general aspect, the polypeptide library disclosed herein is an antibody library, preferably a feline antibody library. Preferably, the antibody library is a synthetic antibody library, more preferably a fully synthetic antibody library.

[0031] The feline antibodies contained within the feline antibody libraries disclosed herein may preferably be single chain Fv (scFv) fragments, Fab fragments, F(ab)2 fragments comprising the VL polypeptide members, and / or VH polypeptide members, or VL / VH polypeptide member combinations described herein.

[0032] In particular, preferred VL / VH polypeptide member combinations showed high expression levels as scFv fragments and Fab fragments.

[0033] Thus, the present invention relates to a polypeptide library comprising at least one scFv, wherein the at least one scFv comprises any of a VH FW1-CDR1-FW2-CDR2-FW3 segment, a VL FW1-CDR1-FW2-CDR2-FW3 segment, or a VL / VH polypeptide member combination described herein. In these embodiments, the VL FW1-CDR1-FW2-CDR2-FW3 segment and the VH FW1-CDR1-FW2-CDR2-FW3 segment, or the VL / VH polypeptide member combination, are contained within a polypeptide member which further comprises a VL CDR3-FW4 segment and a VH CDR3-FW4 segment C-terminal to the respective VL FW1-CDR1-FW2-CDR2-FW3 segment and VH FW1-CDR1-FW2-CDR2-FW3 segment.

[0034] Thus, in a further embodiment, the present invention relates to a polypeptide library comprising a VL / VH polypeptide member combination as disclosed herein in the form of a Fab antibody fragment. More specifically, the VL polypeptide members comprise a VL FW1-FW3 polypeptide sequence in a VL CDR1-FW2-CDR2-FW3-CDR3-FW4 polypeptide sequence with a C-terminally linked constant light chain sequence and a VL FW1-FW3 polypeptide sequence in a VH CDR1-FW2-CDR2-FW3-CDR3-FW4 polypeptide sequence with a C-terminally linked CH1 constant heavy chain sequence. The corresponding library is a Fab antibody library.

[0035] The CDR3 in a VH polypeptide, HCDR3, and the CDR3 in a VL polypeptide, LCDR3, are the major paratopes forming the CDRs in an antibody, which contribute significantly to antigen binding and recognition.

[0036] In our analysis, the most frequent HCDR3 lengths are 11 and 12, each occurring in about 16% (Figure 5). For clarity, the present disclosure refers to the numbering scheme shown in Table 9 and Figure 9, starting the counting at position 98 after the preferred CAR motif. For clarity, the present disclosure refers to the numbering scheme shown in Table 9 and Figure 9, starting the counting at position 98 after the preferred CAR motif. These results are very consistent with recently published data, in which the average amino acid length for HCDR3 was found to be 13.84 ± 3.55 and 13.37 ± 3.38 (counting starting after the C in the CAR motif) for two individual cat samples. In contrast to the high variability of HCDR3 length distribution, the light chain CDRs are much more constrained. The most frequent LCDR3 length observed was 11 amino acids for the lambda light chain, which constituted more than 50% of all analyzed sequences (Figure 5).

[0037] To provide a library suitable for identifying binding agents with diversified binding properties to different antigens, the library of the present invention is diversified in the CDR3 region. For the seven most favorable VH / VL combinations, the LCDR3 and HCDR3 regions were replaced by highly diversified LCDR3 and HCDR3 library modules, respectively. Restriction sites were implemented in the VH and VL sequences to allow library module insertion. For affinity maturation and framework diversification, the library of the present invention further allows combinatorial diversification of HCDR1 and HCDR2.

[0038] The design of the HCDR3 is based on in silico analysis performed on naturally occurring antibody sequences. We chose the CAR motif preceding the HCDR3 because it represents the predominant pattern. Also, the length distribution was chosen to correspond to the natural distribution. The HCDR3 diversity module contains HCDR3 lengths of 7 to 14 amino acids, representing the majority of all lengths identified in our NGS dataset. In certain embodiments of the library, very short or long sequences that frequently show problems with specificity or stability are excluded. Interestingly, for clinical stage human therapeutic antibodies, the CDR3 length according to the IMGT definition had a median length of 12 compared to 15 for the naive human antibody sequence space (Raybould et al. 2019). If the same is true for feline antibodies developed as therapeutics, this is encompassed within the present library design. The amino acid frequency at each position is also based on our NGS dataset. Since each length has a slightly adapted amino acid composition, this was also taken into account in the HCDR3 design. A similar approach was used to design and construct the lambda LCDR3 diversity module, which includes lengths of 9 to 12 amino acids and represents the majority of all lengths in our NGS dataset. For framework regions, critical PTMs were removed within the CDR3 region. In summary, as used herein, the term "LCDR3 diversity module" refers to a variety of different LCDR3 polypeptide segments, or different nucleotide segments encoding such polypeptide segments, having partially randomized amino acid sequences at positions 91 to 99 (optionally including positions 97a, 97b, 97c). In partially randomized amino acid sequences, certain types of amino acids can be excluded at specific positions, and certain amino acids can be found at certain positions with a higher probability than other types of amino acids.

[0039] Thus, in one aspect of the invention, an HCDR3 introduced by an HCDR3 diversity module and contained within a polypeptide library of the invention and having a length of 7-9 amino acids may have the amino acid distribution shown in FIG. 11, an HCDR3 contained within the present library and having a length of 10-12 amino acids may have the amino acid distribution shown in FIG. 12, and an HCDR3 contained within the present library and having a length of 13-14 amino acids may have the amino acid distribution shown in FIG. 13. In summary, as used herein, the term "HCDR3 diversity module" refers to a variety of different HCDR3 polypeptide segments having partially randomized amino acid sequences at positions 96 to 104 (optionally including positions 101a, 101b, 101c, 101d, 101e, 101f, 101g), or different nucleotide segments encoding such polypeptide segments. In the partially randomized amino acid sequences, certain types of amino acids may be excluded at specific positions, and certain amino acids may be found at certain positions with a higher probability than other types of amino acids.

[0040] Furthermore, in one embodiment of the present invention, the LCDR3 introduced by the LCDR3 diversity module and contained in the polypeptide library of the present invention may have the design shown in Table 10. Preferably, the LCDR having a length of 9 to 12 amino acids and contained in the polypeptide library of the present invention may have the amino acid distribution shown in Figure 14.

[0041] According to one aspect of the present invention, diversification of HCDR1 and HCDR2 for improved affinity maturation is achieved by introducing a VH maturation module that randomly or partially randomly comprises HCDR1 and HCDR2 in a VH HCDR1-FW2-HCDR2 segment. The VH maturation module preferably has the design shown in FIG. 15. The "VH maturation module" may also be referred to herein as "HCDR1 / 2 maturation module". In summary, as used herein, the term "HCDR1 / 2 maturation module" refers to various different VH HCDR1-FW2-HCDR2 polypeptide segments with partially randomized amino acid sequences at positions 30 to 35 and positions 49 to 60, or different nucleotide segments encoding such polypeptide segments. In the partially randomized amino acid sequences, certain types of amino acids may be excluded at specific positions, and certain amino acids may be found at certain positions with a higher probability than other types of amino acids.

[0042] When HCDR3 or LCDR3 diversity modules, or VH maturation modules are introduced into the library, they replace the corresponding segments in the VL FW1-CDR1-FW2-CDR2-FW3 and VH FW1-CDR1-FW2-CDR2-FW3 segments, as disclosed above.

[0043] Prior to library generation, any undesirable post-translational modification (PTM) sites were removed from the antibody framework sequences to further optimize the expression and biophysical properties of each VH or VL gene and the corresponding protein. It is important to recognize that post-translational modifications occur in antibody formulations for therapeutic use. Although these may not be an issue in antibody samples produced for initial in vitro characterization, post-translational modifications may occur in antibody samples at high concentrations, under long-term storage conditions, and in vivo. Thus, PTMs may interfere with antibody stability and / or homogeneity, resulting in loss of antibody functionality. Examples of PTMs include, but are not limited to, oxidation (Met, Trp, His), deamidation (Asn, Gln), isomerization (Asp), or N-linked glycosylation (Asn).

[0044] The described library designs provide a variety of advantageous properties, particularly with respect to the VL / VH polypeptide member combinations contained within the library.

[0045] In one embodiment of the disclosure, the polypeptide library provided comprises VL / VH polypeptide member combinations, each VL / VH combination being efficiently displayed on phage, the degree of display can be determined by ELISA and Western blot analysis.

[0046] In another embodiment, substantially all of the VL / VH polypeptide member combinations contained within a library of the invention are expressed in E. coli in a Fab format having a monomer content of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.

[0047] The VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations contained within a polypeptide library according to the invention may be sufficiently expressed in bacterial culture at levels of greater than about 1 mg / L, greater than about 5 mg / L, or greater than about 10 mg / L, or in a mammalian system in an IgG format at levels of greater than about 10 mg / L, greater than about 15 mg / L, or greater than about 20 mg / L.

[0048] Furthermore, in certain embodiments of the invention, all or substantially all of the VL / VH polypeptide member combinations contained within a library may be thermally stable, which may be characterized by a Tm of 60°C or greater or a Tm of 62°C or greater, preferably a Tm of 64°C or greater, more preferably a Tm of 66°C or greater, even more preferably a Tm of 68°C or greater, and most preferably a Tm of 70°C or greater.

[0049] In a further aspect, the present invention relates to a collection of nucleic acid molecules encoding the above-described polypeptide libraries.

[0050] In certain embodiments, the present invention relates to a collection of vectors comprising the above-described nucleic acid molecules. Furthermore, the present invention relates to recombinant host cells comprising the nucleic acid molecules and / or vectors disclosed herein.

[0051] The libraries according to the invention can be used in a method for isolating binding agents specific for an antigen, the method comprising the steps of: (a) contacting a library disclosed herein with an antigen; (b) removing library members that do not bind to the antigen; (c) recovering the library members that bind to the antigen.

[0052] Thus, in a more general aspect, the present invention relates to a feline antibody or antibody fragment comprising a VL polypeptide member and / or a VH polypeptide member or a VL / VH polypeptide member combination as disclosed herein. Preferably, the feline antibody is a complete antibody, a single chain Fv (scFv) fragment, a Fab fragment, or a F(ab)2 fragment.

[0053] In a further aspect, the present invention provides a method for generating a polypeptide library comprising the steps of: (i) providing feline cDNA generated from antibody-expressing tissue, preferably splenocytes, lymphocytes, or bone marrow; (ii) amplifying the VL and VH sequences, preferably by using at least one forward primer selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, and at least one reverse primer selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; (iii) generating VL / VH combination sequences by randomly ligating the amplicons obtained in step ii).

[0054] The method may include various additional steps including and / or required for expression of the VL / VH combination sequences as scFv fragments or soluble Fab fragments, determining the expression levels, and determining the sequence of the best expressing sequence.

[0055] In summary, the present invention provides a feline antibody library based on stable, naturally occurring VH and VL combinations, which VH and VL combinations contain CDR3 regions that mimic the natural length and amino acid distribution found in cats.

[0056] Methods for selecting candidates from synthetic feline antibody libraries include displaying antibody fragments in either scFv or Fab format on phage, bacteria, or yeast. In the preferred system, phage display, antibody fragments are displayed on the surface of bacteriophage as fusion proteins. The antibody-displaying phage are then contacted with the target molecule of interest and affinity selection is performed. Antigens can be displayed on the phage in different forms, for example on the surface of a microtiter plate, in solution on cells, or immobilized on the surface of cells. Phage carrying specific antibody fragments recognize and bind to the corresponding antigen. In contrast, weak or non-specific antibody fragments fail to bind, and the phage are washed away during a selection process commonly referred to as biopanning. After elution of the bacteria and infection of the bacteria with the bacteriophage, the genetic information of the antibody fragments can be transferred to an expression vector and used for screening and sequencing purposes. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 shows an alignment of feline heavy chain FW1-3 sequences of clones with good expression in both scFv and Fab formats. [Diagram 2] Shown is an alignment of feline light chain FW1-3 sequences of clones with good expression in both scFv and Fab formats. [Diagram 3] Figure 1 shows the consensus sequence based on alignment of feline heavy chain FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 sequences of clones with good expression in both scFv and Fab formats. [Figure 4] Figure 1 shows the consensus sequence based on alignment of feline light chain FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 sequences of clones with good expression in both scFv and Fab formats. [Diagram 5] Length distribution analysis of feline LCDR3 and HCDR3. [Figure 6]The results of confirming the expression of scFv fragments having randomly paired natural VL chains and VH chains are shown. [Figure 7] The results of confirming the expression of Fab fragments having randomly paired natural VL and VH chains are shown. [Figure 8] Correlation of expression levels of feline antibody fragments in scFv and Fab formats. [Figure 9] Schematic diagram of VH library building blocks for seven exemplary antibody scaffolds (comprising FW1-3 regions from SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231). HCDR1, HCDR2, and HCDR3 regions (Xaa defines a random amino acid, where Xaa can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V), restriction sites MfeI and XhoI essentially flanking the VH region, NheI and PstI for introduction of HCDR1 / 2 maturation modules, and BssHII in combination with XhoI for introduction of HCDR3 diversity are shown. [Figure 10] Schematic diagram of VL library building blocks for seven exemplary antibody scaffolds (comprising FW1-3 regions from SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224). The LCDR3 region (Xaa defines a random amino acid, Xaa can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V) is shown, as well as the restriction sites BbsI and KpnI for introduction of the LCDR3 diversity module. [Figure 11] The amino acid composition of HCDR3 diversification modules with lengths of 7 to 9 amino acids is shown. [Figure 12] 1 shows the amino acid composition of HCDR3 diversification modules with a length of 10-12 amino acids. [Figure 13] 1 shows the amino acid composition of HCDR3 diversification modules with lengths of 13 and 14 amino acids. [Figure 14]1 shows the amino acid composition of LCDR3 diversification modules with lengths of 9 to 12 amino acids. [Figure 15] 1 shows a schematic concept and amino acid distribution of the HCDR1 / 2 maturation module. The adjacent region 5' to the HCDR1 region, the diversified HCDR1, FW2, the diversified HCDR2, and the following part of FW3 are shown. No length variation is realized in this module. Xaa defines a random amino acid (Xaa can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). FW2 is conserved for all library members. [Figure 16] Screening results after test panning on eGFP are shown in dot blot visualization.

[0058] definition Unless otherwise defined, 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.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0060] "About" in the context of quantity values ​​refers to a mean deviation of up to ±20%, preferably ±10%, most preferably ±5% based on the indicated value. For example, an amount of about 20 mg / ml refers to 20 mg / ml ±6 mg / ml, preferably 20 mg / ml ±4 mg / ml, most preferably 20 mg / ml ±2 mg / ml. It also includes the value itself without any deviation.

[0061] All ranges described herein in the Summary and Description of the Invention include all numbers or values ​​about the numbers in the range, or between the numbers in the range. The ranges of the invention expressly state and describe all integer, decimal, and fractional values ​​within the range. The term "about" may be used to describe the range.

[0062] As used herein, the term "antibody" or "polypeptide binding agent" includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions"), or single chains thereof. A naturally occurring "antibody" is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of an IgG, IgA, or IgD antibody is composed of three domains, CH1, CH2, and CH3, while the heavy chain of an IgM and IgE antibody is composed of four domains, CH1, CH2, CH3, and CH4. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FWs). Each VH and VL is composed of three CDRs and four FWs arranged in the following order from amino-terminus to carboxy-terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. A polypeptide comprising FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4 of the variable regions of the heavy and light chains may be referred to as a "VH or VL polypeptide". The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions 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) via the Fc receptor and the first component (C1q) of the classical complement system. Amino acids are indicated in single letter code.

[0063] The extent of the framework regions and CDRs have been precisely defined for human antibodies (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 of the constituent light and heavy chains, serve to position and align the CDRs that are primarily responsible for binding to the antigen. However, although feline antibodies can be partially aligned with human antibodies, the above numbering scheme is not ideally suited to describe the amino acid positions within the antibody heavy or light chain sequence. In the present invention, the following numbering scheme is used: The antibody heavy chain is defined as VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4. Framework 1 (FW1) is composed of 29 amino acids (X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29). HCDR1 is 6 amino acids long and is defined by positions X30 to X35 (X30, X31, X32, X33, X34, X35). Framework 2 (FW2) is 13 amino acids long and is defined by positions X36 to X48 (X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48). HCDR2 is defined by positions X49 to X60 and has one length variation at position 52, where for clarity, position X52 may be empty or may have an amino acid (X49, X50, X51, X52, X52a, X53, X54, X55, X56, X57, X58, X59, X60). Framework 3 (FW3) is defined by positions X61 to X97 and is 37 amino acids long. HCDR3 is defined by positions X98 to X104 (X98, X99, X100, X101, X101a, X101b, X101c, X101d, X101e, X101f, X101g, X102, X103, X104). This CDR is variable in length, which is indicated by positions X101a to X101g, which may or may not carry an amino acid. For clarity, if one of these positions is empty, the subsequent positions up to X102 are also empty. This concept is also shown in Table 9. Framework 4 (FW4) is defined by positions X105 to X115 (X105, X106, X107, X108, X109, X111, X112, X113, X114, X115). The general idea of ​​the numbering scheme is also shown in FIG.

[0064] The antibody light chain is defined as VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4. Framework 1 (FW1) is composed of 22 amino acids (Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y2). LCDR1 is 6 amino acids long and is defined by positions 30-35 (X30, X31, X32, X33, X34, X35). Framework 2 (FW2) defined by positions 23-29 (Y23, Y24, Y24a, Y24b, Y24c, Y24d, Y24e, Y24f, Y25, Y26, Y27, Y28, Y29). This CDR is variable in length, as indicated by positions Y24a-Y24f, which may or may not carry an amino acid. For clarity, if one of these positions is empty, the subsequent positions up to Y25 are also empty. LCDR2 is defined at positions 30-44d, with length variations at positions Y44a-Y44d, where for clarity positions Y44a-Y44d may be empty or have a four amino acid extension (Y30, Y31, Y32, Y33, Y34, Y35, Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y44a, Y44b, Y44c, Y44d). Framework 3 (FW3) is defined by positions Y55 to Y83 (Y55, Y56, Y57, Y58, Y59, Y60, Y61, Y61a, Y61b, Y62, Y63, Y64, Y65, Y66, Y67, Y68, Y69, Y70, Y71, Y72, Y73, Y74, Y75, Y76, Y77, Y78, Y79, Y80, Y81, Y82, Y83). LCDR3 is defined by positions Y84 to Y92 (Y84, Y85, Y86, Y87, Y88, Y89, Y90, Y90a, Y90b, Y90c, Y91, Y92). This CDR has a length variation, which is indicated by positions Y90a to Y90c, which may or may not carry an amino acid. For clarity, if one of these positions is empty, the subsequent positions up to Y91 are also empty. This concept is also illustrated in Table 10.Framework 4 (FW4) is defined by positions Y93 to Y102 (Y93, Y94, Y95, Y96, Y97, Y98, Y99, Y100, Y101, Y102). The general idea of ​​the numbering scheme is also shown in FIG.

[0065] The terms "antigen-binding portion" or "fragment" of an antibody are used equivalently 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 can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single-domain antibody (dAb) fragment consisting of the VH domain (Ward et al., 1989 Nature 341:544-546); and an isolated complementarity determining region (CDR). A preferred antigen-binding portion or fragment of an antibody is a Fab fragment. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic peptide linker, which allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv), see, for example, Bird et al., 1988 Science 242:423-426, and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies contain 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 the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136).Antigen-binding portions of antibodies can be grafted onto a polypeptide scaffold such as, for example, fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen-binding portions can be incorporated into single-chain molecules that comprise a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides (Zapata et al., 1995 Protein Eng. 8(10)1 057-1062, and U.S. Pat. No. 5,641,870).

[0066] As used herein, the term "feline antibody" refers to an antibody having a variable region in which both the framework and CDR regions are derived from sequences of feline origin. For example, both the framework and CDR regions may be derived from sequences of feline origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a feline sequence, e.g., a feline germline sequence, or a mutated version of a feline germline sequence. The feline antibody of the invention may include amino acid residues not encoded by feline sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0067] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0068] The term "library" refers to a non-naturally occurring collection of different molecules, typically about 10 3 Over 10 pieces, about 10 4 Over 10 pieces, about 10 5 Over 10 pieces, about 10 6 Over 10 pieces, about 10 7 Over 10 pieces, about 10 8 Over 10 pieces, about 10 9 More than, or even about 10 10"Non-naturally occurring" refers to a collection of molecules that includes more than one member. Although different molecules that are "non-naturally occurring" may exist in nature, the entire collection of molecules that establish the library according to the invention does not naturally exist in natural, unengineered organisms. A library in the context of the present invention is a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, each with a single polypeptide or nucleic acid sequence. The sequence differences between the library members are responsible for the diversity present in the library. The library may be in the form of a simple mixture of polypeptides or nucleic acids, or in the form of organisms or cells, such as bacteria, viruses, phages, animal or plant cells, that have been transformed with a library of nucleic acids. Preferably, each individual organism or cell contains only one library member or a limited number of library members. Advantageously, the nucleic acid is incorporated into an expression vector to allow expression of the polypeptide encoded by the nucleic acid. In certain embodiments, the library may be in the form of a population of host organisms, each organism containing one or more copies of an expression vector that contains a single member of the library in nucleic acid form, which 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. The term "collection" is used essentially interchangeably with the term "library."

[0069] Antibody libraries can be derived from immunoglobulins or fragments thereof that are biased towards a certain specificity present in immunized animals or naturally immunized or infected humans. Alternatively, antibody libraries can be derived from naive immunoglobulins or fragments thereof, i.e., immunoglobulins that are not biased towards specificities found in the immune system. Such libraries are referred to as "unbiased" libraries. In a preferred embodiment, the present disclosure provides unbiased antibody libraries, i.e., the libraries have not been pre-exposed to the antigen of interest. Due to the absence of any bias, such libraries contain antibodies that bind to any potential target antigen of interest.

[0070] Typically, immune antibody libraries are constructed with VH and VL gene pools cloned from source B cells by PCR-based (or related) cloning techniques. It is also possible to generate unbiased naive antibody libraries in the same manner. However, unbiased naive antibody libraries can also be generated in a synthetic manner, in which the entire library is constructed entirely in vitro. Recombinant DNA technology can be used to mimic the natural bias and redundancy of natural antibody repertoires. Such antibody libraries are referred to as "synthetic" antibody libraries. The term "fully synthetic" library refers to an antibody library that is completely, i.e., completely de novo, constructed by DNA synthesis, e.g., by total gene synthesis, PCR-based methods, or related DNA technology. In such libraries, the entire DNA is constructed de novo, i.e., the portions encoding the CDRs of the antibodies of the library and the portions encoding the surrounding portions of the CDRs (e.g., framework regions) are constructed de novo. Thus, the terms "synthetic" and "fully synthetic" refer to a new source of DNA. In contrast, in "semi-synthetic" antibody libraries, only parts of the antibodies in the library are constructed de novo, while other parts, e.g., certain CDR regions, are derived from natural sources (there are numerous reviews on this subject, see, e.g., Sidhu et al.; Nat Chem Biol (2006), 2, 682-8).

[0071] In certain embodiments, the present disclosure provides synthetic antibody libraries, preferably semi-synthetic or fully synthetic libraries. In the most preferred embodiments, the present disclosure provides fully synthetic feline antibody libraries.

[0072] The term "germline" refers to complete germline sequences, as well as germline sequences that have been modified or engineered to have minor variations in the amino acid sequence for purposes of, for example, removing undesirable post-translational modification (PTM) sites, removing undesirable cysteines, optimizing the antibody (e.g., affinity, half-life), or introducing desired restriction sites, or modifications resulting from errors in synthesis, amplification, or cloning.

[0073] The term "VL / VH polypeptide member combination" refers to a combination (pairing) of one VL polypeptide sequence and one VH polypeptide sequence. The pairing may be between different domains of one polypeptide chain comprising a VL polypeptide sequence and a VH polypeptide sequence, as in, for example, an scFv, or may be between two polypeptide chains comprising a VL polypeptide sequence and a VH polypeptide sequence, as in, for example, an Fab fragment.

[0074] The term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked plasmids or plasmids contained within liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0075] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been subsequently modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids are commonly identified herein according to the known one-letter or three-letter amino acid codes.

[0076] 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 corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0077] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity, over a specified region, or, when not specified, over the entire sequence), 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, the 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. Thus, a "substantially identical sequence" according to the present invention can include any of the disclosed sequences with 1 or 2 or 3 or 4 or 5 amino acid exchanges, preferably 1-3, more preferably 1 or 2 amino acid exchanges. Preferably, the exchanges can be conservative amino acid exchange(s).

[0078] For sequence comparison, typically, one sequence serves as a reference sequence, to which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence relative to reference sequence based on the programmed parameters.

[0079] 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 such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in successive generations, due either to 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.

[0080] 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, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in 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. Certain vectors are also capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors that serve equivalent functions, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

[0081] The term "expression system" refers to a host cell and a compatible expression vector under suitable conditions, e.g., conditions suitable for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells (e.g., BL21, TGF1-) and plasmid vectors, insect host cells and mammalian host cells (e.g., HEK239 cells or CHO cells) and vectors.

[0082] As used herein, the term "substantially all" means that the component to which it refers is more or less pure. Only small amounts of other different components are present that do not limit or affect the advantageous properties of the component. Depending on the nature of the component, essentially 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 this component.

[0083] As used herein, the term "randomized amino acid sequence" refers to a sequence in which the amino acid at any position in a given sequence can be any amino acid, preferably any naturally occurring amino acid. As used herein, "partially randomized amino acid sequence" refers to a sequence in which any position in a given sequence can be any amino acid, except one or more specifically excluded amino acids. Furthermore, in a "partially randomized amino acid sequence", some positions in a given sequence can contain specifically defined amino acids. Randomized and partially randomized amino acid sequences can also be referred to as "randomly diversified". Thus, a "randomly diversified" CDR is characterized by a randomized or partially randomized amino acid sequence. In a partially randomized amino acid sequence, a certain type of amino acid can be excluded at a specific position, and a certain amino acid can be found at a certain position with a higher probability than another type of amino acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] In a first aspect, the invention provides a polypeptide library comprising a VL polypeptide member comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, The present invention relates to a polypeptide library comprising at least one sequence selected from SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136 and / or SEQ ID NO:137.

[0085] Preferably, at least one sequence comprised within a VL FW1-CDR1-FW2-CDR2-FW3 segment comprised within a VL polypeptide is selected from SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, and / or SEQ ID NO:135.

[0086] Preferably, the polypeptide library comprises at least 2, at least 5, at least 10, at least 15, or preferably all, of the VL polypeptide members.

[0087] In a second aspect, the present invention provides a polypeptide library comprising VH polypeptide members comprising a FR1-CDR1-FW2-CDR2-FR3 segment, wherein the FR1-CDR1-FW2-CDR2-FR3 segment is selected from the group consisting of SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO: No. 166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:202.

[0088] In a preferred embodiment, the present invention relates to a polypeptide library comprising VL polypeptide members comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment comprises at least one sequence selected from SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224, and / or SEQ ID NO:224; and / or to a polypeptide library comprising VH polypeptide members comprising a VH FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VH FW1-CDR1-FW2-CDR2-FW3 segment comprises at least one sequence selected from SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231.

[0089] Preferably, the polypeptide library comprises at least 2, at least 5, at least 10, at least 15, or preferably all, of the VH polypeptide members.

[0090] Within the context of the present invention, a "FW1-CDR1-FW2-CDR2-FW3 segment" is a polypeptide comprising, from N- to C-terminus, at least framework region 1, followed by CDR1, followed by framework region 2, followed by CDR2, followed by framework region 3 of the variable region of a heavy or light chain of a feline antibody. Such a VH polypeptide member comprising a VH FW1-CDR1-FW2-CDR2-FW3 segment of the variable region of a heavy chain of a feline antibody is also referred to as a "VH FW1-FW3 polypeptide", and a VL polypeptide member comprising a VH FW1-CDR1-FW2-CDR2-FW3 segment of the variable region of a light chain of a feline antibody is also referred to herein as a "VL FW1-FW3 polypeptide".

[0091] In a further main aspect, the present invention provides a polypeptide library comprising VH polypeptide members comprising at least one VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, the sequence being - FW1 is X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, - HCDR1 is X30, X31, X32, X33, X34, or X35; - FW2 is X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, - HCDR2 is X49, X50, X51, X52, X52a, X53, X54, X55, X56, X57, X58, X59, X60, - FW3 is X61, X62, X63, X64, X65, X66, X67, X68, X69, X70, X71, X72, X73, X74, X75, X76, X77, X78, X79, X80, X81, X82, X83, X84, X85, X86, X87, X88, X89, X90, X91, X92, X93, X94, X95, X96, X97, - HCDR3 is X98, X99, X100, X101, X101a, X101b, X101c, X101d, X101e, X101f, X101g, X102, X103, X104, - FW4 includes X105, X106, X107, X108, X109, X111, X112, X113, X114, and X115; X1 denotes a first amino acid position, X2 denotes a second amino acid position, and so on, and the nature of the amino acids at corresponding positions are defined as follows: [Table C-1] [Table C-2] (Amino acids in bold and underlined are preferred amino acids, and "-" defines an empty position).

[0092] Thus, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence: (D / Q / Y / E)(V / E)(Q / L / R)LV(E / Q)SGGD(L / R)V(K / Q)PGGSLRL(T / I / A)C(V / M)(A / G)SGF(T / P / I / N)(F / V / L )XaaXaaXaaXaaXaaXaaWVRQ(A / T)PG(K / M)GLQWV (I / V)S(R / K)D(N / D)(A / P / V)(K / R / M / E)NTL(Y / L)LQM(N / T / D)(S / G / N)LKTED(T / A / M)ATYYC(A / S / T)(R / K / N / T / G)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWG (Q / R) The amino acids of the HCDRs are designated as variable amino acids (Xaa), which can be any one of A (Ala), R (Arg), N (Asn), D (Asp), C (Cys), Q (Gln), E (Glu), G (Gly), H (His), I (Ile), L (Leu), K (Lys), M (Met), F (Phe), P (Pro), S (Ser), T (Thr), W (Trp), Y (Tyr), or V (Val).

[0093] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence: (D / Q / Y / E)(V / E)(Q / L / R)LV(E / Q)SGGD(L / R)V(K / Q)PGGSLRL(T / I / A)C(V / M)(A / G)SGF(T / P / I / N)(F / V / L)(S / R / G / T / N)(S / R / G / N / D / L)(Y / H / F / D)(G / W / D / E / A / S / Y)M(S / N / T / D / H)WVRQ(A / T)PG(K / M)GLQWV(A / S / T)(A / S / Y / G / T / Q / D)(I / N / L)(S / G / R / D / A)(G / F / Y / S / N / D / R)(S / T / D / G / N)G(G / S / I / H / D / A)(S / A / T / V / N)(T / I)(Y / A / G / N / H)Y(A / S / G / I)DSVKGRFT(I / V)S(R / K)D(N / D)(A / P / V)(K / R / M / E)NTL(Y / L)LQM(N / T / D)(S / G / N)LKTED(T / A / M)ATYYC(A / S / T)(R / K / N / T / G)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWG(Q / R)G(A / T / V)(L / P)VTVSS (SEQ ID NO: 233) may be included, The amino acids of HCDR 1 and 2 are defined in the sequence.

[0094] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment has the following sequence, DVQLVESGGDLVKPGGSLRLTCVASGFTFXaaXaaXaaXaaXaaXaaWVRQAPGKGLQWVXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaDSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCARXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWGQGALVTVSS (SEQ ID NO: 234), or DVQLVESGGDLVKPGGSLRLTCVASGFTFSSYGMSWVRQAPGKGLQWVAAISGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCARXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWGQGALVTVSS (sequence number 235).

[0095] The polypeptide library according to the present invention comprises at least about 1.0*10 9 In another embodiment, the present invention provides a method for the preparation of a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprising at least about 1.0*10 10 , at least about 1.0*10 11 of at least about 1.0*10 12 of, or at least about 1.0*10 13 A polypeptide library is provided, comprising different VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segments.

[0096] The VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 consensus sequence with alternative amino acids is shown diagrammatically in FIG.

[0097] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment is selected from the group consisting of DEQLVESGGDLVKPGGSLRLTCVASGFPF (SEQ ID NO: 240), DEQLVESGGDLVKPGGSLRLTCVASGFTL (SEQ ID NO: 241), DEQLVESGGDLVKPGGSLRLTCVGSGFTF (SEQ ID NO: 242), DVQLVESGGDLVKPGGSLRLACVASGFTF (SEQ ID NO: 243), DVQLVESGGDLVKPGGSLRLICVASGFTF (SEQ ID NO: 244), DVQLVESGGDLVKPGGSLRLTCVASGFIF (SEQ ID NO: 245), DVQLVESGGDLVKPGGSLRLTCVASGF PF (SEQ ID NO: 246), DVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 247), DVQVVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 248), DVRLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 249), DVRLVESGGDRVKPGGSLRLTCMASGFNV (SEQ ID NO: 250), EVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 251), QVLLVQSGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 252), or YVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 253).

[0098] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises a FW1 sequence selected from DVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 247), EVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 251), or DVQLVESGGDLVQPGGSLRLTCVASGFTF (SEQ ID NO: 405).

[0099] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises an HCDR1 sequence selected from GSYDMT (SEQ ID NO:254), NNFAMS (SEQ ID NO:255), NSYAMS (SEQ ID NO:256), RGYAMT (SEQ ID NO:257), RSHWMN (SEQ ID NO:258), SDYDMS (SEQ ID NO:259), SGYSMN (SEQ ID NO:260), SLYDMS (SEQ ID NO:261), SNYDMS (SEQ ID NO:262), SNYGMD (SEQ ID NO:263), SNYGMS (SEQ ID NO:264), SRYGMS (SEQ ID NO:265), SSYAMS (SEQ ID NO:266), SSYEMN (SEQ ID NO:267), SSYGMS (SEQ ID NO:268), SSYYMH (SEQ ID NO:269), TGDAMS (SEQ ID NO:270), or TNYAMS (SEQ ID NO:271).

[0100] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises an HCDR1 sequence selected from SDYDMS (SEQ ID NO: 259), SNYGMS (SEQ ID NO: 264), SNYDMS (SEQ ID NO: 262), SRYGMS (SEQ ID NO: 265), SSYEMN (SEQ ID NO: 267), SSYYMH (SEQ ID NO: 269), or NSYAMS.

[0101] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises a FW2 sequence selected from WVRQAPGKGLQWV (SEQ ID NO: 271), WVRQAPGMGLQWV (SEQ ID NO: 273), WVRQAPGRGLQWV (SEQ ID NO: 274), or WVRQTPGKGLQWV (SEQ ID NO: 275).

[0102] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises the FW2 sequence of WVRQAPGKGLQWV (SEQ ID NO: 271).

[0103] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment is selected from the group consisting of DSVKGRFTISKDDAENTLYLQMNSLKTEDTATYYCAG (SEQ ID NO: 298), DSVKGRFTISRDNAENTLLLQMNSLKTEDTATYYCAR (SEQ ID NO: 299), DSVKGRFTISRDNAKNTLSLQMDSLKTEDTATYYCAT (SEQ ID NO: 300), DSVKGRFTISRDNAKNTLYLQMDSLKTEDTATYYCTS (SEQ ID NO: 301), DSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR (SEQ ID NO: 302), DSVKGRFTISRDNAKNTLYLQMNNLKTEDTATYYCAG (SEQ ID NO: 303), DSVKGRFTISRDNAKNTLYLQMNSLKTEDMATYYCAR (SEQ ID NO: 304), DSVKGRFTISRD The FW3 sequence is selected from NAKNTLYLQMNSLKTEDTATYYCAK (SEQ ID NO: 305), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 306), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAT (SEQ ID NO: 307), DSVKGRFTISRDNAMNTLYLQMNSLKTEDAATYYCAR (SEQ ID NO: 308), DSVKGRFTISRDNARNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 309), DSVKGRFTISRDNPKNTLYLQMTSLKTEDTATYYCAR (SEQ ID NO: 310), DSVKGRFTISRDNVKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 311), or DSVKGRFTVSRDNAKNTLYLQMNSLKTEDTATYYCSR (SEQ ID NO: 312).

[0104] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises a FW3 sequence selected from DSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR (SEQ ID NO: 302), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAK (SEQ ID NO: 305), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAN (SEQ ID NO: 414), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 306), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAT. (SEQ ID NO: 307).

[0105] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment is selected from the group consisting of AAIAYNGGNTGYA (SEQ ID NO: 276), AAIGHDGSTTAYA (SEQ ID NO: 277), AAIRGSGGVTYYA (SEQ ID NO: 278), AAISGSGDSTYYA (SEQ ID NO: 279), AAISYNGGGTGYS (SEQ ID NO: 280), AANSGTGSSTYYA (SEQ ID NO: 281), ADISGSGGATAYA (SEQ ID NO: 282), AGISGSGITTYYA (SEQ ID NO: 283), AGISTSGGNTYYA (SEQ ID NO: 284), AGITSGGNTYYA (SEQ ID NO: 285), AQISDSGGSTY and comprising an HCDR2 sequence selected from YA (SEQ ID NO:286), AYIDNDGSSTYYA (SEQ ID NO:287), AYIRYDGNTIHYG (SEQ ID NO:288), AYIRYDGSSTNYA (SEQ ID NO:289), AYISSGGSTYYA (SEQ ID NO:290), CAIGGTGSRTLYA (SEQ ID NO:291), SAISFDGSGTGYA (SEQ ID NO:292), SALSESGHSTIYA (SEQ ID NO:293), SSISSGGTTYYA (SEQ ID NO:294), STIDSGGNTHYI (SEQ ID NO:295), TDISRSGATTYYA (SEQ ID NO:296), or TTISGSGGSTYYA (SEQ ID NO:297).

[0106] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises a HCDR2 sequence selected from AAISYNGGGTGYS (SEQ ID NO: 280), AANSGTGSSTYYA (SEQ ID NO: 281), AAIAYNGGNTGYA (SEQ ID NO: 276), AAISGSGDSTYYA (SEQ ID NO: 279), AYISSGGSTYYA (SEQ ID NO: 290), AQISDSGGSTYYA (SEQ ID NO: 286), or ADISGSGGATAYA (SEQ ID NO: 282).

[0107] Preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises a FW4 sequence selected from WGQGALVTVSS, WGQGAPVTVSS, WGQGTLVTVSS, WGQGVLVTVSS, or WGRGALVTVSS.

[0108] More preferably, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises the FW4 sequence WGQGALVTVSS.

[0109] For the purpose of construct integration, introduction of restriction sites, removal of PTM sites, etc., FW1, HCDR1, FW2, HCDR2, FW3, HCDR3, FW4 of the above VH may be modified to the following sequences: The VH FW1 sequence can be DVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 247), DVQLVESGGDLVQPGGSLRLTCVASGFTF (SEQ ID NO: 405), and / or DVQLVQSGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 406).

[0110] The HCDR1 sequence can be SDYDMS (SEQ ID NO: 259), SNYGMS (SEQ ID NO: 264), SNYDMS (SEQ ID NO: 262), SRYGMS (SEQ ID NO: 265), SSYEMN (SEQ ID NO: 267), SSYYMH (SEQ ID NO: 269), and / or SSYAMS (SEQ ID NO: 266).

[0111] The VH FW2 sequence can be WVRQAPGKGLQWV (SEQ ID NO:271).

[0112] The HCDR2 sequence can be AAISYNGGGTGYS (SEQ ID NO:280), AANSGTGSSTYYA (SEQ ID NO:281), AAIAYNGGNTGYA (SEQ ID NO:276), AAISGSGDSTYYA (SEQ ID NO:279), AYISSGGSTYYA (SEQ ID NO:290), AQISDSGGSTYYA (SEQ ID NO:286), and / or ADISGSGGATAYA (SEQ ID NO:282).

[0113] The VH FW3 sequence can be DSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR (SEQ ID NO: 302), or DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 306).

[0114] The VH FW4 sequence can be WGQGALVTVSS.

[0115] FW1, HCDR1, FW2, HCDR2, FW3, HCDR3 and FW4 of the above VH may be combined.

[0116] In a further main aspect, the present invention provides a polypeptide library comprising VL polypeptide members comprising at least one VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, the sequence being: - FW1 is Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22; - LCDR1 is Y23 Y24, Y24a, Y24b, Y24c, Y24d, Y24e, Y24f, Y25, Y26, Y27, Y28, Y29, - FW2 is Y30, Y31, Y32, Y33, Y34, Y35, Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y44a, Y44b, Y44c, Y44d; - LCDR2 is Y45, Y46, Y47, Y48 Y49, Y50, Y51, Y52, Y53, Y54, - FW3 is Y55, Y56, Y57, Y58, Y59, Y60, Y61, Y61a, Y61b, Y62, Y63, Y64, Y65, Y66, Y67, Y68, Y69, Y70, Y71, Y72, Y73, Y74, Y75, Y76, Y77, Y78, Y79, Y80, Y81, Y82, Y83; - HCDR3 is Y84, Y85, Y86, Y87, Y88, Y89, Y90, Y90a, Y90b, Y90c, Y91, Y92, - FW4 is Y93, Y94, Y95, Y96, Y97, Y98, Y99, Y100, Y101, Y102, For a polypeptide library, Y1 denotes a first amino acid position, Y2 denotes a second amino acid position, and so on, and the nature of the amino acids at corresponding positions are defined as follows: [Table D-1] [Table D-2] (Amino acids in bold and underlined are preferred amino acids, and "-" defines an empty position)

[0117] Thus, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following amino acid sequence for the LCDR: Q(S / P / E)(V / G)(P / L / V)(T / N)Q(P / E)(P / S / T)S(V / L / M)S(G / A / T)(A / G / S / T)( L / P)G(Q / G / T)(R / A / T / K)(V / I)(T / R)(I / M / L)(S / T)CXaaXaaXaaXaaXaaXaaXa aXaaXaaXaaXaaXaaXaaXaaW(Y / V)(Q / R)Q(I / V / L / K / H)(P / S)(G / R / Y)(M / K / R / Q / T / S)(A / P / R)(P / F)(K / Q / S)(T / L / A / V / Y)(L / I / V / F)(I / V / L)(Y / V / G / H)(- / Y)(- / Y)(- / S)(- / D)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(D / A / N / S)R(F / I / L / V)SGS(K / L)(- / D)(- / A)(S / V)(G / A)(S / N / T / Q / I / A)(T / K / A)(G / A)(S / T / L / V) L(T / A / L)(I / V)(T / S)G(L / P / A)Q(A / S / T / P)EDE(A / G / V)(D / N)Y(Y / H)CXaaXaa XaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(F / I)G(G / R)GTH(L / V)(T / S)VL(Sequence number 236) Xaa can be any one of A (Ala), R (Arg), N (Asn), D (Asp), C (Cys), Q (Gln), E (Glu), G (Gly), H (His), I (Ile), L (Leu), K (Lys), M (Met), F (Phe), P (Pro), S (Ser), T (Thr), W (Trp), Y (Tyr), or V (Val) (the variable amino acids are designated as Xaa).

[0118] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following sequence: Q(S / P / E)(V / G)(P / L / V)(T / N)Q(P / E)(P / S / T)S(V / L / M)S(G / A / T)(A / G / S / T)(L / P)G(Q / G / T)(R / A / T / K) (V / I)(T / R)(I / M / L)(S / T)C(T / A / S / G)(G / S / L)(S / T / -)(S / A / G / T / R / -)(S / N / G / P / E / -)(N / A / F / V / -)(I / N / V / S / -)(G / T / V / -)(V / R / I / S / T / G / A / -)(G / V / A / D / S / T)(N / D / G / T / S / M / V / K / Y)(Y / S / G / -)(V / A / I)(S / N / G / D / Y)W(Y / V)(Q / R)Q(I / V / L / K / H)(P / S)(G / R / Y)(M / K / R / Q / T / S)(A / P / R)(P / F)(K / Q / S)(T / L / A / V / Y) (L / I / V / F)(I / V / L)(Y / V / G / H)(- / Y)(- / Y)(- / S)(- / D)(G / A / S / D / E / R / Y)(N / S / D / T)(S / N / T / D / R / K / F)( N / R / K / D / E / Y / H)(R / Q / L)(P / N / G)(S / P)(G / S / E)VP(D / A / N / S)R(F / I / L / V)SGS(K / L)(- / D)(- / A)(S / V)(G / A)(S / N / T / Q / I / A)(T / K / A)(G / A)(S / T / L / V)L(T / A / L)(I / V)(T / S)G(L / P / A)Q(A / S / T / P)EDE(A / G / V)(D / N)Y(Y / H)CXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(F / I)G(G / R)GTH(L / V)(T / S)VL (SEQ ID NO: 237); The amino acids of LCDR 1 and 2 are defined in the sequence.

[0119] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment has the following sequence: QSVPTQPPSVSGALGQRVTISCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa XaaXaaXaaXaaDRFSGSKSGSTGSLTITGLQAEDEADYYCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaFGGGTHLTVL (SEQ ID NO: 238), or QSVPTQPPSVSGALGQRVTISCTGSSSNIGVGNYVSWYQQIPGMAPKTLIYGNSNRPSGVPDRFSGSKSGSTGSLTITGLQAEDEADYYCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaFGGGTHLTVL (SEQ ID NO: 239), It may have the structure: QSGPNQPSSVSGALGQRVTISCTGSSSNIGRGNYVSWYQQVPGMAPKTLIYGNSNRPSGVPDRFSGSSKSGSTGSLTITGLQAEDEADYYCXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaFGGGTHLTVL (sequence number 416).

[0120] The polypeptide library according to the present invention comprises at least about 1.0*10 9 In another embodiment, the present invention provides a method for the preparation of a 1.0*10 LCDR fragment according to SEQ ID NO: 236, 237, 238, 239, or 416, comprising administering to the patient a 1.0*10 LCDR fragment according to SEQ ID NO: 236, 237, 238, 239, or 416, comprising administering to the patient a 1.0*10 LCDR fragment according to SEQ ID NO: 236, 237, 238, 239, or 416, and ... 10 , at least about 1.0*10 11 of at least about 1.0*10 12 of, or at least about 1.0*10 13 A polypeptide library is provided, comprising different VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segments.

[0121] The VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 consensus sequence with alternative amino acids is shown diagrammatically in FIG.

[0122] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is QSVVTQPPSVSGALGQAVTISC (SEQ ID NO: 313), QSVLTQPPSVSGALGQTVTISC (SEQ ID NO: 314), QSVLTQPPSVSGGLGQKVTISC (SEQ ID NO: 315), QPVVTQPPSVSGALGQTVTISC (SEQ ID NO: 316), QSVLTQPPSMSGALGQTVTISC (SEQ ID NO: 317), QPVLTQPPSVSGSLGQRVTISC (SEQ ID NO: 318), QSGPNQPSSVSGALGQRVTISC (SEQ ID NO: 319), QSGPNQPSSVSGALGQRVTM and QPVVTQPPSVSGALGQRVTISC (SEQ ID NO: 326), QPVLTQPPSVSGALGQRVTISC (SEQ ID NO: 327), or QPVVTQPPSLSGSLGATARLTC (SEQ ID NO: 328).

[0123] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a FW1 sequence selected from (SEQ ID NO: 318), QSGPNQPSSVSGALGQRVTISC (SEQ ID NO: 319), QSGPNQPSSVSGTLGQTITISC (SEQ ID NO: 322), QSVVTQPPSVSGALGQRVTISC (SEQ ID NO: 324), and / or QPVLTQPPSVSGALGQRVTISC (SEQ ID NO: 325).

[0124] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is selected from the group consisting of AGSANNIGITDVN (SEQ ID NO: 329), AGSGSNIGIVDVN (SEQ ID NO: 330), AGSGSNIGIAGVN (SEQ ID NO: 331), AGSANNNGIVGVN (SEQ ID NO: 332), AGSGSNIGIVAGVN (SEQ ID NO: 333), TGTSSNIGSGNYVS (SEQ ID NO: 334), TGIDTYVG (SEQ ID NO: 335), TGVDTYVD (SEQ ID NO: 336), TGVDSYVG (SEQ ID NO: 337), SGAGSYVA (SEQ ID NO: 338), and a LCDR1 sequence selected from TGAGSYVG (SEQ ID NO: 339), TGVGNYVD (SEQ ID NO: 340), TGVGMSVD (SEQ ID NO: 341), TGAGYVG (SEQ ID NO: 342), TVGSVFVD (SEQ ID NO: 343), GSSTGAVTTSNFAS (SEQ ID NO: 344), TGSSSNVGRGNYVS (SEQ ID NO: 345), TGSSPNIGRGNYVS (SEQ ID NO: 346), TGSSSNIGRGNYVS (SEQ ID NO: 347), TLSSGFNVGGYYIS (SEQ ID NO: 348), or TLSREVSVGVKGIY (SEQ ID NO: 349).

[0125] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a LCDR1 sequence selected from TGTSSNIGSGNYVS (SEQ ID NO: 334), TGIDTYVG (SEQ ID NO: 335), TGAGSYVG (SEQ ID NO: 339), TGVGNYVD (SEQ ID NO: 340), TVGSVFVD (SEQ ID NO: 343), TGSSSNVGRGNYVS (SEQ ID NO: 345), and / or TGSSSNIGRGNYVS (SEQ ID NO: 347).

[0126] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is selected from the group consisting of WYQQLPGKAPKLLIY (SEQ ID NO: 350), WYQQLPGKAPSLLIY (SEQ ID NO: 351), WYQQHPRKAPKLLIY (SEQ ID NO: 352), WYQQLPGKAPQLLIY (SEQ ID NO: 353), WYQQLPGKAPKALIY (SEQ ID NO: 354), WYRQVPGIAPSLLIY (SEQ ID NO: 355), WFQQIPGMAPKTIIV (SEQ ID NO: 356), WYQQIPGMAPKTIIY (SEQ ID NO: 357), WYQQIPGRAPKTIIY (SEQ ID NO: 358), WYQQIPGRAPKTIIY (SEQ ID NO: 359), WYQQIPGRAPKTIIY (SEQ ID NO: 360), WYQQIPGRAPKTIIY (SEQ ID NO: 361), WYQQIPGRAPKTIIY (SEQ ID NO: 362), WYQQIPGRAPKTIIY (SEQ ID NO: 363), WYQQIPGRAPKTIIY (SEQ ID NO: 364), WYQQIPGRAPKTIIY (SEQ ID NO: 365), WYQQIPGRAPKTIIY (SEQ ID NO: 366), WYQQIPGRAPKTIIY (SEQ ID NO: 367), WYQQIPGRAPKTIIY (SEQ ID NO: 368), WYQQIPGRAPKTIIY (SEQ ID NO: 369), WYQQIPGRAPKTIIY (SEQ ID NO: 370), WYQQIPGRAPKTIIY (SEQ ID NO: 371), WYQQIP sequence number 358), WYQQVPGMAPKTIIY (SEQ ID NO: 359), WYQQIPGMAPKTVIY (SEQ ID NO: 360), WVQQKPYQRFQGLVG (SEQ ID NO: 361), WYQQLSGTPPKLLIY (SEQ ID NO: 362), WYQQLSGTAPKVLIY (SEQ ID NO: 363), WYQQLSGTAPKLLIY (SEQ ID NO: 364), WYQQVSGTAPKLLIY (SEQ ID NO: 365), WFQQKPGSPPRYLLYYYSD (SEQ ID NO: 366), or WYQQKPGSPPRYFLHYYSD (SEQ ID NO: 367).

[0127] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a FW2 sequence selected from WYRQVPGIAPSLLIY (SEQ ID NO: 355), WFQQIPGMAPKTIIV (SEQ ID NO: 356), WYQQVPGMAPKTIIY (SEQ ID NO: 359), WYQQIPGMAPKTVIY (SEQ ID NO: 360), WYQQLSGTPPKLLIY (SEQ ID NO: 362), and / or WYQQVSGTAPKLLIY (SEQ ID NO: 365).

[0128] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is selected from the group consisting of ANNRRPSSVP (SEQ ID NO: 368), ASDRRPSGVP (SEQ ID NO: 369), GSSNRPSGVP (SEQ ID NO: 370), ASTRRPSGVP (SEQ ID NO: 371), ENFKRPSGVP (SEQ ID NO: 372), GNTNRPSGVP (SEQ ID NO: 373), YDSNRPSGVP (SEQ ID NO: 374), DNSNRPSGVP (SEQ ID NO: 375), DDNKRPSGVP (SEQ ID NO: 376). , GNRYRPSGVP (SEQ ID NO: 377, RNSNRLSEVP (SEQ ID NO: 378), GNSYRPSGVP (SEQ ID NO: 379), RNTNRLSEVP (SEQ ID NO: 380), GTSYRNPGVP (SEQ ID NO: 381), GNSDRLSGVP (SEQ ID NO: 382), GDKNRPSGVP (SEQ ID NO: 383), GDTNRPSGVP (SEQ ID NO: 384), SDKHQGPGVP (SEQ ID NO: 385), or STNELGPGVP (SEQ ID NO: 386).

[0129] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a LCDR2 sequence selected from ENFKRPSGVP (SEQ ID NO: 372), GNTNRPSGVP (SEQ ID NO: 373), DDNKRPSGVP (SEQ ID NO: 376), GNRYRPSGVP (SEQ ID NO: 377), RNTNRLSEVP (SEQ ID NO: 380), GNTDRLSGVP (SEQ ID NO: 410), and / or GDTNRPSGVP (SEQ ID NO: 384).

[0130] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is selected from the group consisting of DRFSGSKSGATGSLTISGLQAEDEADYYC (SEQ ID NO: 387), ERISGSRSGNTGSLTITGLQTEDEADYYC (SEQ ID NO: 388), DRFSGSKSGNTGSLTITGLQAEDEADYYC (SEQ ID NO: 389), DRFSGSKSGNTGSLTITGLQAEDEADYYC (SEQ ID NO: 390), ERLSGSKSGNTGSLTITGLQPEDEADYYC (SEQ ID NO: 391), DRFSGSKSGSSGSLTITGLQADDEVDYYC (SEQ ID NO: 392), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 393), DRFSGSKSGSTGTLAITGLQAEDEGDYYC (SEQ ID NO: 394), DRFSGSKSGNTGTLTITGLQAEDEADY YC (SEQ ID NO: 395), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 396), ARFSGSKSGSTGTLTITGLQAEDEANYYC (SEQ ID NO: 397), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 398), NRFSGSKSGSTATLTITGLQAEDEADYYC (SEQ ID NO: 399), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 400), DRFSGSKSGITATLTITGLQAEDEADYYC (SEQ ID NO: 401), ARFSGSLVGQKAVLTITGAQSEDEAEYYC (SEQ ID NO: 402), DRFSGSKSGTTGSLTITGLQTEDEADYYC (SEQ ID NO: 403), or DRFSGSKSGSTGSLTITGLQAEDEADYYC (SEQ ID NO: 404).

[0131] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a FW3 sequence selected from DRFSGSKSGSSGSLTITGLQADDEVDYYC (SEQ ID NO: 392), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 396), ARFSGSKSGSTGTLTITGLQAEDEANYYC (SEQ ID NO: 397), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 400), DRFSGSKSGITATLTITGLQAEDEADYYC (SEQ ID NO: 401), DRFSGSKSGTTGSLTITGLQTEDEADYYC (SEQ ID NO: 403), or DRFSGSKSGSTGSLTITGLQADDEADYYC. (SEQ ID NO: 415).

[0132] Preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises a FW4 sequence selected from FGGGTHLTVL (SEQ ID NO:208), FGGGTHLSVL (SEQ ID NO:209), FGRGTHLTVL (SEQ ID NO:210), FGGGTHVTVL (SEQ ID NO:211), or IGGGTHVTVL (SEQ ID NO:212).

[0133] More preferably, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises the FW4 sequence of FGGGTHLTVL (SEQ ID NO: 208).

[0134] For the purpose of construct integration, introduction of restriction sites, removal of PTM sites, etc., FW1, HCDR1, FW2, HCDR2, FW3, HCDR3, FW4 of the above VLs may be modified to the following sequences: VL FW1 can be (SEQ ID NO: 318), QSGPNQPSSVSGALGQRVTISC (SEQ ID NO: 319), QSGPNQPSSVSGTLGQTITISC (SEQ ID NO: 322), QSVVTQPPSVSGALGQRVTISC (SEQ ID NO: 324), and / or QPVLTQPPSVSGALGQRVTISC (SEQ ID NO: 325).

[0135] HCDR1 can be TGTSSNIGSGNYVS (SEQ ID NO: 334), TGIDTYVG (SEQ ID NO: 335), TGAGSYVG (SEQ ID NO: 339), TGVGNYVD (SEQ ID NO: 340), TVGSVFVD (SEQ ID NO: 343), TGSSSNVGRGNYVS (SEQ ID NO: 345), and / or TGSSSNIGRGNYVS (SEQ ID NO: 347).

[0136] VL FW2 can be WYRQVPGIAPSLLIY (SEQ ID NO: 355), WFQQIPGMAPKTIIV (SEQ ID NO: 356), WYQQVPGMAPKTIIY (SEQ ID NO: 359), WYQQIPGMAPKTVIY (SEQ ID NO: 360), WYQQLSGTPPKLLIY (SEQ ID NO: 362), and / or WYQQVSGTAPKLLIY (SEQ ID NO: 365).

[0137] LCDR2 can be (SEQ ID NO:372), GNTNRPSGVP (SEQ ID NO:373), DDNKRPSGVP (SEQ ID NO:376), GNRYRPSGVP (SEQ ID NO:377), RNTNRLSEVP (SEQ ID NO:380), GNTDRLSGVP (SEQ ID NO:410), and / or GDTNRPSGVP (SEQ ID NO:384).

[0138] VL FW3 can be DRFSGSKSGSSGSLTITGLQAEDEVDYYC (SEQ ID NO: 411), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 396), ARFSGSKSGSTGTLTITGLQAEDEANYYC (SEQ ID NO: 397), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 400), DRFSGSKSGITATLTITGLQAEDEADYYC (SEQ ID NO: 401), DRFSGSKSGTTGSLTITGLQTEDEADYYC (SEQ ID NO: 403), and / or DRFSGSKSGSTGSLTITGLQAEDEADYYC (SEQ ID NO: 404).

[0139] VL FW 4 can be FGGGTHLTVL (SEQ ID NO: 208).

[0140] Preferably, at least one sequence comprised within a VL FW1-CDR1-FW2-CDR2-FW3 segment or a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprised within a VL polypeptide is selected from SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, and / or SEQ ID NO:224.

[0141] Preferably, at least one sequence comprised within a VH FW1-CDR1-FW2-CDR2-FW3 segment or a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprised within a VH polypeptide is selected from: SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230 and / or SEQ ID NO:231.

[0142] It has been found that the variable heavy and variable light chains comprising polypeptide members comprising the VH FW1-CDR1-FW2-CDR2-FW3 and VL FW1-CDR1-FW2-CDR2-FW3, and / or VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 and VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segments of the present invention are compatible with phage display and show good expression levels as scFV and Fab fragments, and therefore represent an ideal scaffold for constructing synthetic feline antibody polypeptide libraries with pre-selected and tested framework regions.

[0143] A further important aspect of the present invention relates to a polypeptide library comprising at least one VL / VH polypeptide member combination, wherein the at least one VL / VH polypeptide member combination comprises at least one VL polypeptide member as defined above and at least one VH polypeptide member as defined above. Preferably, the library comprises at least one VL polypeptide member comprising a sequence selected from SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, more preferably SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, and / or SEQ ID NO:224. and at least a VH polypeptide member comprising a sequence selected from SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, more preferably SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231. Preferably, the library comprises at least two, at least five, at least ten, at least fifteen, or preferably all, of said VL and VH polypeptide members.

[0144] 1. A polypeptide library comprising at least one VL / VH polypeptide member combination, wherein at least one VL / VH polypeptide member combination is selected from the group consisting of SEQ ID NO:236 and SEQ ID NO:232, SEQ ID NO:237 and SEQ ID NO:233, SEQ ID NO:238 and SEQ ID NO:234, SEQ ID NO:239 and SEQ ID NO:235, more preferably SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:77 and SEQ ID NO:142, SEQ ID NO:78 and SEQ ID NO:143, 3, SEQ ID NO:79 and SEQ ID NO:144, SEQ ID NO:80 and SEQ ID NO:145, SEQ ID NO:81 and SEQ ID NO:146, SEQ ID NO:82 and SEQ ID NO:147, SEQ ID NO:83 and SEQ ID NO:148, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:87 and SEQ ID NO:152, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:89 and SEQ ID NO:154, SEQ ID NO:90 and SEQ ID NO:155, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:93 and SEQ ID NO:158, SEQ ID NO:94 and and SEQ ID NO:159, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:96 and SEQ ID NO:161, SEQ ID NO:97 and SEQ ID NO:162, SEQ ID NO:98 and SEQ ID NO:163, SEQ ID NO:99 and SEQ ID NO:164, SEQ ID NO:100 and SEQ ID NO:165, SEQ ID NO:101 and SEQ ID NO:166, SEQ ID NO:102 and SEQ ID NO:167, SEQ ID NO:103 and SEQ ID NO:168, SEQ ID NO:104 and SEQ ID NO:169, SEQ ID NO:105 and SEQ ID NO:170, SEQ ID NO:106 and SEQ ID NO:171, SEQ ID NO:107 and SEQ ID NO:172, SEQ ID NO:108 and SEQ ID NO:173, SEQ ID NO:10 9 and SEQ ID NO:174, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:111 and SEQ ID NO:176, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:113 and SEQ ID NO:178, SEQ ID NO:114 and SEQ ID NO:179, SEQ ID NO:115 and SEQ ID NO:180, SEQ ID NO:116 and SEQ ID NO:181, SEQ ID NO:117 and SEQ ID NO:182, SEQ ID NO:118 and SEQ ID NO:183, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:120 and SEQ ID NO:185, SEQ ID NO:121 and SEQ ID NO:186, SEQ ID NO:122 and SEQ ID NO:187, SEQ ID NO:123 and SEQ ID NO:188,23. A polypeptide library comprising sequence combinations selected from SEQ ID NO:124 and SEQ ID NO:189, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:126 and SEQ ID NO:191, SEQ ID NO:127 and SEQ ID NO:192, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:130 and SEQ ID NO:195, SEQ ID NO:131 and SEQ ID NO:196, SEQ ID NO:132 and SEQ ID NO:197, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:136 and SEQ ID NO:201, SEQ ID NO:137 and SEQ ID NO:202, SEQ ID NO:218 and SEQ ID NO:225, SEQ ID NO:219 and SEQ ID NO:226, SEQ ID NO:220 and SEQ ID NO:227, SEQ ID NO:221 and SEQ ID NO:228, SEQ ID NO:222 and SEQ ID NO:229, SEQ ID NO:223 and SEQ ID NO:230, and / or SEQ ID NO:224 and SEQ ID NO:230. Preferably, the polypeptide library comprises at least two, at least five, at least ten, at least fifteen, or preferably all, of the VL / VH polypeptide members.

[0145] Preferably, the polypeptide library comprises at least one VL / VH polypeptide member combination, the at least one VL / VH polypeptide member combination being selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:180 and SEQ ID NO:182, SEQ ID NO:183 and SEQ ID NO:184, SEQ ID NO:185 and SEQ ID NO:186, SEQ ID NO:187 and SEQ ID NO:189, SEQ ID NO:190 and SEQ ID NO:192, SEQ ID NO:193 and SEQ ID NO:194, SEQ ID NO:195 and SEQ ID NO:196, SEQ ID NO:197 and SEQ ID NO:198, SEQ ID NO:199 and SEQ ID NO:200, SEQ ID NO:201 and SEQ ID NO:202, SEQ ID NO:203 and SEQ ID NO:204, SEQ ID NO:205 and SEQ ID NO:206, SEQ ID NO:207 and SEQ ID NO:208, SEQ ID NO:209 and SEQ ID NO:210 and 112 and SEQ ID NO:177, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:218 and SEQ ID NO:225, SEQ ID NO:219 and SEQ ID NO:226, SEQ ID NO:220 and SEQ ID NO:227, SEQ ID NO:221 and SEQ ID NO:228, SEQ ID NO:222 and SEQ ID NO:229, SEQ ID NO:223 and SEQ ID NO:230, and / or SEQ ID NO:224 and / or SEQ ID NO:230. Preferably, the polypeptide library comprises at least two, at least five, at least ten, at least fifteen, or preferably all, of said VL / VH polypeptide members.

[0146] The described VL / VH polypeptide member combinations may be included in the polypeptide library in a form in which each of the VL FW1-FW3 polypeptide sequences and each of the VH FW1-FW3 polypeptide sequences of a particular VL / VH polypeptide member combination are contained within a single polypeptide molecule, and in which the VL FW1-FW3 polypeptide sequence is located at the N-terminus of the VH FW1-FW3 polypeptide sequence. Thus, VL / VH polypeptide member combinations may be included in the polypeptide library in a form such that, for each VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment polypeptide sequence and each VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment polypeptide sequence, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment polypeptide sequence is positioned N-terminal to the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment polypeptide sequence.

[0147] In a highly preferred embodiment, the polypeptide comprising the VL / VH polypeptide member combination may preferably be an scFv. Thus, the present invention relates to a polypeptide library comprising at least one scFv, wherein at least one scFv comprises any of the VH FW1-CDR1-FW2-CDR2-FW3 segment, the VL FW1-CDR1-FW2-CDR2-FW3 segment, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, or the VL / VH polypeptide member combination described herein. The corresponding library may be referred to as a "scFv antibody library". In these embodiments, the VL FW1-CDR1-FW2-CDR2-FW3 segment and the VH FW1-CDR1-FW2-CDR2-FW3 segment, or VL / VH polypeptide member combination, are comprised within a polypeptide member which further comprises a VL CDR3-FR4 segment and a VH CDR3-FR4 segment at the C-terminus of the respective VL FW1-CDR1-FW2-CDR2-FW3 segment and VH FW1-CDR1-FW2-CDR2-FW3 segment. Specifically, the VL / VH polypeptide member combination is comprised within the library in the form of the polypeptide VL(FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4)-linker-VH(FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4).

[0148] In an alternative embodiment, the described VL / VH polypeptide member combinations may be included within a polypeptide library in the form of two polypeptides, where the VL polypeptide comprises a VL FW1-FW3 and / or a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment polypeptide sequence and comprises an antibody constant light chain sequence C-terminal to the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment polypeptide sequence, and the VH polypeptide comprises a VH FW1-FW3 and / or a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 polypeptide sequence and comprises an antibody CH1 constant heavy chain sequence C-terminal to the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment polypeptide sequence. Preferably, the VL and VH polypeptides are linked by a disulfide bridge between the constant light chain sequence and the CH1 constant heavy chain sequence. Alternatively, the VL and VH polypeptides may be linked by a linker. The VL / VH polypeptide member combination may preferably be a Fab antibody fragment. More specifically, the VL polypeptide comprises a VL FW1-FW3 polypeptide sequence in a VL CDR1-FW2-CDR2-FW3-CDR3-FR4 polypeptide with a C-terminal linked constant light chain and a VH FW1-FW3 polypeptide sequence in a VH CDR1-FW2-CDR2-FW3-CDR3-FR4 polypeptide with a C-terminal linked C1 constant heavy chain sequence.

[0149] Thus, in a further embodiment, the present invention relates to a polypeptide library comprising at least one Fab antibody fragment, wherein the at least one Fab antibody fragment comprises any of a VH FW1-CDR1-FW2-CDR2-FW3 segment, a VL FW1-CDR1-FW2-CDR2-FW3 segment, a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, or a VL / VH polypeptide member combination as described herein. Corresponding libraries may be referred to as "Fab antibody libraries".

[0150] A further aspect of the present invention relates to a library as described herein comprising a polypeptide member or a combination of polypeptide members, wherein the polypeptide member or combination of polypeptide members comprises a sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to the sequences disclosed above. Such sequences may also be referred to as substantially identical sequences.

[0151] In a specific embodiment of an scFv library, the library comprises at least one scFv fragment, wherein the VH polypeptide member is N-terminally linked to the VL polypeptide member by a linker of about 15 to about 25, preferably about 16 to about 21, more preferably about 17 to about 19, and most preferably about 18 amino acids. In a preferred embodiment, the linker comprises the amino acids GGSSRSSSSGGGGSGGGG (SEQ ID NO: 207).

[0152] In further embodiments of the scFv library, the VL / VH polypeptide member combination may comprise an N- or C-terminal affinity tag, preferably a FLAG tag. The affinity tag facilitates isolation and purification of the scFv antibody fragments. In further embodiments of the scFv library, the VL / VH polypeptide member combination may comprise a full-length or truncated phage pIII polypeptide sequence linked to a VH polypeptide. Preferably, the VL / VH polypeptide member combination comprises a FLAG tag linked N-terminally to the VL polypeptide sequence and a phage pIII polypeptide linked C-terminally to the VH polypeptide sequence.

[0153] In the above embodiments, the VH polypeptide further comprises a feline variable heavy chain framework 4 region (FW4 region). The VH FW4 region is comprised within the VH polypeptide C-terminal to the FW3 sequence. In a preferred embodiment, at least one VH polypeptide comprises a FW4 region whose sequence is selected from the amino acid sequence WGQGALVTVSS (SEQ ID NO: 213), WGQGAPVTVSS" (SEQ ID NO: 214), WGQGTLVTVSS (SEQ ID NO: 215) WGQGVLVTVSS (SEQ ID NO: 216), and WGRGALVTVSS (SEQ ID NO: 217). In a most preferred embodiment, one VH polypeptide comprises a FW4 region sequence selected from the amino acid sequence WGQGALVTVSS (SEQ ID NO: 213).

[0154] In the above embodiment, the VL polypeptide further comprises a feline variable light chain framework 4 region (FW4 region). The VL FW4 region is comprised within the VL polypeptide C-terminal to the FW3 sequence. In a preferred embodiment, at least one VL polypeptide comprises a FW4 region sequence selected from the amino acid sequence FGGGTHLTVL (SEQ ID NO: 208), FGGGTHLSVL (SEQ ID NO: 209), FGRGTHLTVL (SEQ ID NO: 210), FGGGTHVTVL (SEQ ID NO: 211), and IGGGTHVTVL (SEQ ID NO: 212). In a most preferred embodiment, one VL polypeptide comprises a FW4 region sequence selected from the amino acid sequence FGGGTHLTVL (SEQ ID NO: 208).

[0155] In the above embodiment, the different VL and VH polypeptide members comprise different feline-specific CDR3 polypeptide sequences (LCDR3 in the VL and HCDR3 in the VH polypeptide) between the FW3 and FW4 segments.

[0156] Within the context of the present invention, a "feline-specific CDR3 polypeptide sequence" is a CDR3 polypeptide sequence that is naturally expressed in a feline, or a synthetic sequence based on a naturally occurring sequence.

[0157] In a preferred embodiment, the CDR3 polypeptide sequence comprises a randomized or partially randomized amino acid sequence.

[0158] In certain embodiments, the disclosure provides a polypeptide library comprising HCDR3s, the HCDR3s covering more than 50% of the naturally occurring HCDR3 lengths of the feline HCDR3 repertoire. In other aspects, the polypeptide library comprises more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the naturally occurring HCDR3 lengths of the feline HCDR3 repertoire.

[0159] In certain embodiments, the disclosure provides a polypeptide library, wherein the HCDR3 regions of essentially all members of the library are flanked by unique restriction sites.

[0160] In certain aspects, the disclosure provides a polypeptide library comprising an HCDR3 length of 3 to 16 amino acids according to the numbering scheme shown in Table 9 and Figure 9. Preferably, the HCDR3 contained in the library has a length of 7 to 14 amino acids. More preferably, the HCDR may have an average length of 8 to 13 amino acids or 10 to 13 amino acids, most preferably 10 to 12 amino acids. Preferably, the amino acid sequence of the HCDR3 is fully or partially randomized.

[0161] In certain embodiments, the HCDR3 contained in the polypeptide library according to the present invention comprises a randomly diversified HCDR3. The HCDR3 contained in the library may have the design shown in Table 9. Preferably, the HCDR3 contained in the polypeptide library of the present invention and having a length of 7-9 amino acids may have the amino acid distribution shown in FIG. 11, the HCDR3 contained in the present library and having a length of 10-12 amino acids may have the amino acid distribution shown in FIG. 12, and the HCDR3 contained in the present library and having a length of 13-14 amino acids may have the amino acid distribution shown in FIG. 13. As an example, of the HCDR3 contained in the library and having a length of 13 amino acids and the amino acid distribution shown in FIG. 13, 23% have a D at position 98, 9% have a D at position 99, 9% have a D at position 100, 9% have an E at position 98, 2% have an E at position 99, and so on.

[0162] In certain embodiments, the present disclosure provides a synthetic feline antibody polypeptide library, the HCDR3 region comprising at least about 1.0*10 9 In another aspect, the disclosure provides a synthetic feline antibody polypeptide library having a diversity of at least about 1.0*10 10 , at least about 1.0*10 11 , at least about 1.0*10 12, or at least about 1.0*10 13 The present invention provides a polypeptide library having a diversity of

[0163] The generation of diversified HCDR3 regions is described in Example 7. Polynucleotides encoding the diversified HCDR3 regions described herein may be referred to as "HCDR3 diversity modules."

[0164] In certain embodiments, the present invention provides a polypeptide library, wherein the LCDR3 regions of essentially all members of the library are flanked by unique restriction sites.

[0165] In certain embodiments, the present invention provides a polypeptide library comprising LCDR3s, the LCDR3s covering more than 50% of the naturally occurring LCDR3 lengths of the feline LCDR3 repertoire, in other embodiments, the polypeptide library comprises more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% of the naturally occurring LCDR3 lengths of the feline LCDR3 repertoire.

[0166] In certain aspects, the disclosure provides a polypeptide library comprising an LCDR3 for a VL lambda of length 8 to 13 amino acids according to the numbering scheme shown in Table 10 and Figure 10, preferably 9 to 12 amino acids.

[0167] In an alternative embodiment, the VL may be a kappa light chain.

[0168] In certain embodiments, the LCDR3 comprises a randomly diversified LCDR. The LCDR3 contained in the polypeptide library may have the design shown in Table 10. Preferably, the LCDRs contained in the present polypeptide library and having a length of 9 to 12 amino acids may have the amino acid distribution shown in Figure 14.

[0169] In certain embodiments, the present disclosure provides a synthetic feline antibody polypeptide library, the LCDR3 region comprising at least about 1.0*10 5 In another embodiment, the present invention provides a synthetic feline antibody polypeptide library having a diversity of at least about 11.0*10 6 , at least about 1.0*10 7 , or at least about 1.0*10 8 The present invention provides a polypeptide library having a diversity of

[0170] The generation of diversified LCDR3 regions is described in Example 8. Polynucleotides encoding the diversified LCDR3 regions described herein may be referred to as "LCDR3 diversity modules."

[0171] In certain aspects, the disclosure provides a polypeptide library, comprising the HCDR3 designs shown in Table 9 and Figures 11-13, and the LCDR3 designs shown in Table 10 and Figure 14.

[0172] In a further embodiment, the VH polypeptide sequences contained within the polypeptide library may comprise diversified VH HCDR1-FW2-HCDR2 segments to allow affinity maturation. In the diversified VH HCDR1-FW2-HCDR2 segments according to the invention, HCDR1 preferably has a fixed length of 6 amino acids and HCDR2 preferably has a fixed length of 13 amino acids. The randomly diversified HCDR1 and HCDR2 contained within the VH HCDR1-FW2-HCDR2 segments may have the design shown in Figure 15. Preferably, the diversified FW2 contained within the diversified VH HCDR1-FW2-HCDR2 segments has the sequence WVRQAPGKGLQWV (SEQ ID NO: 241) shown in Figure 15.

[0173] The polypeptide library can include VH polypeptide members as described above, wherein the VH CDR1-FW2-CDR2 of said members is replaced by a diversified VH HCDR1-FW2-HCDR2 segment.

[0174] The generation of diversified VH HCDR1-FW2-HCDR2 segments is described in Example 9. The polynucleotides encoding the diversified VH HCDR1-FW2-HCDR2 segments described herein may be referred to as "VH maturation modules."

[0175] In further embodiments, a polypeptide library can include VL polypeptide members, in which a VL LCDR1-FW2-LCDR2 segment has been replaced with a diversified VL LCDR1-FW2-LCDR2 segment, the diversified VL LCDR1-FW2-LCDR2 segment comprising an LCDR1 and / or LCDR2 having a partially or completely random amino acid sequence. A polynucleotide encoding a diversified VL HCDR1-FW2-HCDR2 segment as described herein can be referred to as a "VL maturation module."

[0176] In further embodiments, the polypeptide members contained within the polypeptide library lack post-translational modification (PTM) sites to optimize the expression and biophysical properties of each VH or VL polypeptide member. PTMs do not necessarily occur in every antibody sample produced, but may occur in antibody samples at high concentrations, under long-term storage conditions, and in vivo. PTMs may interfere with antibody stability and / or homogeneity, resulting in loss of antibody functionality. Examples of PTMs include, but are not limited to, oxidation (Met, Trp, His), deamidation (Asn, Gln), isomerization (Asp), or N-linked glycosylation (Asn). VH or VL polypeptide members may also lack other undesirable sequences. Preferably, the VH or VL polypeptide member comprises a glycosylation motif (comprising the amino acid sequence NxS NxT, where x is not P, e.g., NAS, NCS, ND, S NES, NFS, NGS, NHS, NIS, NKS, NLS, NMS, NNS, NQS, NRS, NSS, NTS, NVS, NWS, NYS, NAT, NCT, NDT, NET, NFT, NGT, NHT, NIT, NKT, NLT, NMT, NNT, NQT, NRT, NST, NTT, NVT, NWT, NYT), an asparagine deamidation motif (comprising the amino acid sequence NG, NS, NT, NH), an aspartate isomerization motif (comprising the amino acid sequence DG DS DT DD DH), a lysine glycosylation motif (comprising the amino acid sequence KE KD KK), an integrin binding aVb3 motif (comprising the amino acid sequence RGD RYD KGD NGR), integrin-binding a4b1 motif (containing the amino acid sequence LDV), integrin-binding a2b1 motif (containing the amino acid sequence DGE), CD11c / CD18-binding motif (containing the amino acid sequence GPR), fragmentation motif (containing the amino acid sequences DP, DQ), and hydrophobic motif (containing the amino acid sequences FF, FW, WW, WF).

[0177] In yet another aspect, the disclosure provides a polypeptide library comprising VL / VH polypeptide member combinations, wherein substantially all of the VL / VH combinations are efficiently displayed on phage, as may be measured by sandwich phage ELISA, as described herein in Example 4, or by Western blot analysis.

[0178] In another embodiment, substantially all of the VL / VH polypeptide member combinations contained within a library of the invention are expressed in E. coli in a Fab format having a monomer content of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.

[0179] In further embodiments, the VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations are fully expressed in an IgG format in a mammalian system. More specifically, the present invention provides a polypeptide library comprising VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations, wherein substantially all of the VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations expressed in an IgG format in a mammalian system have a monomer content of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In certain aspects, the mammalian system comprises cells selected from HKB11 cells, PERC.6 cells, or CHO cells.

[0180] The VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations contained within the polypeptide libraries according to the present invention may be sufficiently expressed in bacterial culture at levels of greater than about 1 mg / L, greater than about 5 mg / L, or greater than about 10 mg / L in bacterial culture.

[0181] The VL and / or VH polypeptide members and / or VL / VH polypeptide member combinations contained within the polypeptide libraries according to the invention may be satisfactorily expressed in a mammalian system in IgG format at levels of greater than about 10 mg / L, greater than about 50 mg / L, or greater than about 100 mg / L, or greater than about 150 mg / L, or greater than about 200 mg / L.

[0182] In certain aspects, the present disclosure provides a polypeptide library, in which all or substantially all VL / VH polypeptide member combinations are thermally stable. Thermal stability can be measured according to commonly known methods by differential scanning fluorimetry, particularly nanodifferential scanning fluorimetry. In certain aspects, the disclosure provides a library, in which essentially all VL / VH polypeptide member combinations have a Tm of 60°C or higher. In further embodiments, substantially all VL / VH polypeptide member combinations contained within the library can have a Tm of 62°C or higher, preferably a Tm of 64°C or higher, more preferably a Tm of 66°C or higher, even more preferably a Tm of 68°C or higher, and most preferably a Tm of 70°C or higher.

[0183] In a further aspect, the present invention relates to a collection of nucleic acid molecules that encode the above-described polypeptide libraries.

[0184] In certain embodiments, the invention provides a collection of nucleic acid molecules encoding a polypeptide library comprising VL polypeptide members comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, No. 111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224, and / or SEQ ID NO:224.

[0185] In certain aspects, the invention relates to a collection of nucleic acid molecules encoding a polypeptide library comprising VL polypeptide members comprising a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, wherein the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment comprises at least one sequence selected from SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, or SEQ ID NO:239.

[0186] In a further aspect, the invention provides a collection of nucleic acid molecules encoding a polypeptide library comprising VH polypeptide members comprising a FW1-CDR1-FW2-CDR2-FW3 segment, wherein the FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, 10. The present invention relates to a collection of nucleic acid molecules comprising at least one sequence selected from SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231.

[0187] In certain aspects, the invention relates to a collection of nucleic acid molecules encoding a polypeptide library comprising VH polypeptide members comprising VH polypeptide members comprising a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, wherein the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment comprises at least one sequence selected from SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, or SEQ ID NO: 235.

[0188] In a further aspect, the invention relates to a collection of nucleic acid molecules encoding a polypeptide library comprising at least one VL / VH polypeptide member combination, wherein the at least one VL / VH polypeptide member combination comprises at least one VL polypeptide member as defined above and at least one VH polypeptide member as defined above. Preferably, the collection of nucleic acid molecules encodes a polypeptide library and is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135. and at least one VL polypeptide member comprising a sequence selected from SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, and / or SEQ ID NO:224, and at least a VH polypeptide member comprising a sequence selected from SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150 SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231.

[0189] In a further preferred embodiment, the present invention relates to a collection of nucleic acid molecules encoding a polypeptide library comprising at least one VL / VH polypeptide member combination, wherein at least one VL / VH polypeptide member combination is selected from the group consisting of SEQ ID NO:236 and SEQ ID NO:232, SEQ ID NO:237 and SEQ ID NO:233, SEQ ID NO:238 and SEQ ID NO:234, SEQ ID NO:239 and SEQ ID NO:235, SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO: No. 77 and SEQ ID NO: 142, No. 78 and SEQ ID NO: 143, No. 79 and SEQ ID NO: 144, No. 80 and SEQ ID NO: 145, No. 81 and SEQ ID NO: 146, No. 82 and SEQ ID NO: 147, No. 83 and SEQ ID NO: 148, No. 84 and SEQ ID NO: 149, No. 85 and SEQ ID NO: 150, No. 86 and SEQ ID NO: 151, No. 87 and SEQ ID NO: 152, No. 88 and SEQ ID NO: 153, No. 89 and SEQ ID NO: 154, No. 90 and SEQ ID NO: 155, No. 91 and SEQ ID NO: 156, No. 92 and SEQ ID NO: Column number 157, SEQ ID NO:93 and SEQ ID NO:158, SEQ ID NO:94 and SEQ ID NO:159, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:96 and SEQ ID NO:161, SEQ ID NO:97 and SEQ ID NO:162, SEQ ID NO:98 and SEQ ID NO:163, SEQ ID NO:99 and SEQ ID NO:164, SEQ ID NO:100 and SEQ ID NO:165, SEQ ID NO:101 and SEQ ID NO:166, SEQ ID NO:102 and SEQ ID NO:167, SEQ ID NO:103 and SEQ ID NO:168, SEQ ID NO:104 and SEQ ID NO:169, SEQ ID NO:105 and SEQ ID NO:170, SEQ ID NO:106 and SEQ ID NO:171, SEQ ID NO:107 and and SEQ ID NO:172, SEQ ID NO:108 and SEQ ID NO:173, SEQ ID NO:109 and SEQ ID NO:174, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:111 and SEQ ID NO:176, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:113 and SEQ ID NO:178, SEQ ID NO:114 and SEQ ID NO:179, SEQ ID NO:115 and SEQ ID NO:180, SEQ ID NO:116 and SEQ ID NO:181, SEQ ID NO:117 and SEQ ID NO:182, SEQ ID NO:118 and SEQ ID NO:183, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:120 and SEQ ID NO:185, SEQ ID NO:121 and SEQ ID NO:186,SEQ ID NO:122 and SEQ ID NO:187, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:124 and SEQ ID NO:189, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:126 and SEQ ID NO:191, SEQ ID NO:127 and SEQ ID NO:192, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:130 and SEQ ID NO:195, SEQ ID NO:131 and SEQ ID NO:196, SEQ ID NO:132 and SEQ ID NO:197, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO: No. 199, SEQ ID NO: 135 and SEQ ID NO: 200, SEQ ID NO: 136 and SEQ ID NO: 201, and / or SEQ ID NO: 137 and SEQ ID NO: 202, SEQ ID NO: 218 and SEQ ID NO: 225, SEQ ID NO: 219 and SEQ ID NO: 226, SEQ ID NO: 220 and SEQ ID NO: 227, SEQ ID NO: 221 and SEQ ID NO: 228, SEQ ID NO: 222 and SEQ ID NO: 229, SEQ ID NO: 223 and SEQ ID NO: 230, and / or SEQ ID NO: 224 and SEQ ID NO: 230.

[0190] In certain embodiments, the present invention relates to a collection of vectors comprising the above-described nucleic acid molecules.Furthermore, the present invention relates to recombinant host cells comprising the nucleic acid molecules and / or vectors disclosed herein.

[0191] In one aspect, the disclosure provides a method for isolating a binding agent specific for an antigen, the method comprising: (a) contacting a library disclosed herein with an antigen; (b) removing library members that do not bind to the antigen; (c) recovering the library members that bind to the antigen.

[0192] Thus, the present invention relates to an antibody or antibody fragment obtained from the polypeptide library described herein. Preferably, the antibody or antibody fragment is obtainable by the method described above.

[0193] In a more general aspect, the present invention relates to a feline antibody or antibody fragment comprising a VL polypeptide member and / or a VH polypeptide member or a VL / VH polypeptide member combination according to the present disclosure. Preferably, the feline antibody is a complete antibody, a single chain Fv (scFv) fragment, a Fab fragment, or a F(ab)2 fragment.

[0194] Antibodies according to the invention may be selected from IgG1a, IgG1b, or IgG2 isotypes. Isotypes may also include modified versions of one of these classes, modifications made to alter Fc function, for example to enhance or reduce effector function or binding to Fc receptors.

[0195] In a further aspect, the present invention provides a method for generating a polypeptide library comprising the steps of: (i) providing feline cDNA generated from antibody-expressing tissue, preferably splenocytes, lymphocytes, or bone marrow; (ii) amplifying the VL and VH sequences, preferably by using at least one forward primer selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, and at least one reverse primer selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, to obtain an amplicon; (iii) generating VL / VH combination sequences by randomly ligating the amplicons obtained in step ii).

[0196] In a preferred embodiment, the primers used in step (ii) are designed to incorporate a secondary primer binding site at the 5' end of the amplified VL amplicon and at the 3' end of the amplified VH amplicon and / or to incorporate a flexible linker at the 3' end of the VL amplicon and at the 5' end of the VH amplicon.

[0197] Preferably, the VL amplicon and the VH amplicon are linked in step (iii) by a linker sequence encoding 15 to 25 amino acids.

[0198] Optionally, the method further comprises the step of joining a sequence encoding an affinity tag to the VL / VH combination sequence obtained in step (iii).

[0199] The method of the invention may optionally further comprise a step (iv) of cloning the VL / VH combination sequence into a vector, preferably selected from a plasmid, a phagemid or an expression cassette.

[0200] The method of the invention may optionally further comprise the step (v) of expressing in a phage expression system a single chain Fv fragment or a Fab fragment comprising a VL / VH combination sequence linked to a full-length or truncated phage pIII polypeptide.

[0201] The method of the present invention may optionally further comprise the step (vi) of isolating the phage expressing the VL / VH combination sequence by affinity tag, and optionally determining the sequence of the VL / VH combination sequence in the isolated phage.

[0202] The method of the present invention may optionally further comprise the step (vii) of infecting a bacterium with the phage isolated in step (iv). Preferably, the bacterium is E. coli.

[0203] The method of the present invention may optionally further comprise the step (viii) of subcloning the VL / VH combination sequence from the isolated phage in step (vii) into an expression system and expressing the VL / VH combination sequence as a soluble scFv fragment or Fab fragment. Preferably, the expression system may be a bacterial expression system.

[0204] The method of the invention may optionally further comprise a step (ix) of determining the expression level of the soluble scFv fragment in the expression system by ELISA.

[0205] The method of the invention may optionally further comprise a step (x) of sequencing DNA encoding the VL / VH combined sequences from high-expressing clones of the expression system.

[0206] The method of the present invention may optionally further comprise the steps of cloning the VL sequence and VH combination sequence determined by sequencing in step (x) into a Fab expression system (xi), optionally expressing the Fab fragment in a cell (xii), and optionally determining the expression level of the soluble Fab fragment in the cell (xiii). In an optional step (xiv), the sequence of the VL and VH combination sequence in the high expressing cell may be determined by sequencing.

[0207] Finally, VL / VH combination sequences that are highly expressed in scFv and Fab format and therefore likely to be stable can be determined in step xv) by comparing the sequences obtained in step (x) and step (xiv).

[0208] In a further embodiment, the method may comprise the step of introducing said LCDR3 diversity module and / or said HCDR3 diversity module into at least one VL and / or VH sequence of the combination sequence identified in the disclosed method, preferably into the sequence identified in step (xv).

[0209] In a further embodiment, the method may comprise the step of introducing said VL maturation module and / or said VH maturation module into the VL and / or VH sequence of a combination sequence identified in the disclosed method, preferably into the sequence identified in step (xv).

[0210] In a further embodiment, step xi) comprises introducing the feline constant light part of the light chain (CL-lambda) and the constant heavy chain region (CH1) into an expression system such that in the expression system the VL polypeptide is expressed C-terminally linked CL-lambda and the VH polypeptide is expressed C-terminally linked CH1.

[0211] Optionally, both cysteines at the C-terminus of CL and the N-terminus of CH1 are mutated to serine.

[0212] In a further aspect, the present invention relates to a polypeptide library or a collection of nucleic acids obtainable or obtainable by the method of the present invention. EXAMPLES

[0213] Example 1: NGS analysis of feline immunoglobulin repertoire For generation of the NGS dataset, primers were designed to PCR amplify VL and VH sequences from feline bone marrow cDNA (Amsbio #CD-704) (modified from Steiniger et al. 2017). Sequences were polyclonally cloned into a pUC19 storage plasmid. For NGS, VL and VH sequences were amplified via PCR from the storage plasmid introducing overhangs for Illumina sequencing (primer sequences are shown in Table 1). [Table 1] TIFF2025508603000010.tif156155

[0214] The NGS data was analyzed using the Geneious software suite, which analyzes the CDR3 length distribution (Table 2) and CDR1-3 amino acid composition. Numbering and CDR definitions were adapted from existing antibody numbering schemes and applied to fit the feline and canine antibody sequences. Examples of identified VL and VH sequences in the context of the synthetic feline library are shown in Figures 1 and 2. In total, 28628 individual VL-lambda and 22445 VH sequences that met certain quality criteria were used for analysis.

[0215] The distribution of HCDR3 lengths of the analyzed feline antibodies shows an average amino acid length of 10.28±2.53, with the most frequent HCDR3 lengths being 11 and 12, each occurring in about 16% (Table 2). For clarity, we refer to the numbering scheme shown in FIG. 9, starting the count at position 98 after the preferred CAR motif. In contrast to the high variability of the HCDR3 length distribution, the light chain CDRs are much more constrained. The most frequent LCDR3 length observed was 11 amino acids for the lambda light chain, which constituted more than 50% of all analyzed sequences (see Table 2 and FIG. 5). [Table 2]

[0216] An analysis of total amino acid usage in cat LCDR3, HCDR1, HCDR2, and HCDR3 was performed. The amino acid variability at a given position in aligned sequences of the same CDR length was calculated as a percentage of all amino acids present at the corresponding position. The amino acid usage for the most common CDR lengths identified in our data set is shown in Tables 3 and 4 (LCDR3: 12 AA, HCDR1: 6 AA, HCDR2: 13 AA, HCDR3: 12 AA). Grey shading indicates the more frequently occurring amino acid at each position. An analysis of sequences with different CDR lengths was performed (data not shown) and the results were taken into account for the design of CDR modules used for synthetic libraries (see Example 6).

[0217] The feline antibody repertoire follows similar trends in amino acid usage as observed in dogs. The high frequency of charged amino acids at the base of the HCDR3 loop is similar to the dog repertoire. Also, the FDY motif at the end of the HCDR3 at positions 99-101 is predominant. However, the HCDR3 is preferentially adjacent to the CAR motif at position 94, in contrast to CAK in dogs. [Table 3-1] [Table 3-2] [Table 4]

[0218] Example 2: Amplification of variable antibody regions from feline bone marrow cDNA and cloning into a phage display vector Based on the NGS analysis in Example 1, primers were designed to PCR amplify a specific set of VL-lambda and VH sequences from feline bone marrow cDNA. Overhangs were designed to i) allow Gibson assembly into the display vector, ii) introduce an N-terminal FLAG tag, and iii) introduce an 18 amino acid linker between VL and VH via overlap extension PCR to generate scFv fragments. The display vector introduced a truncated ct-pIII gene, a trypsin cleavage site before ct-pIII, and an alternative antibiotic resistance (chloramphenicol). For VL-lambda, a set of 10 forward primers and 10 reverse primers were designed. For VH, a set of 12 forward primers and 10 reverse primers were designed. Primers were designed to cover the most frequent VL and VH groups, covering more than 59% of the VL-lambda and more than 64% of the VH sequences identified in the NGS analysis. The concentration on the highly abundant VL and VH groups is based on sequences that are favorable with respect to their biological functionality. [Table 5] TIFF2025508603000016.tif228155 TIFF2025508603000017.tif48155

[0219] Example 3: Display of scFv fragments on phage and anti-FLAG pull-down The scFv fragments cloned into the display vector featuring random combinations of cDNA-derived VL-lambda and VH sequences were produced on the surface of the phage according to standard procedures using VCSM13-based helper phage (see also Example 10). The phage preparations were analyzed by performing colony PCR in infected XL1-Blue E. coli, revealing approximately 85% of the analyzed clones showing fragments with the correct size, validating the overall cloning approach of native VL / VH sequences into the display vector. For enrichment of phage particles displaying intact FLAG-scFv fragments, anti-FLAG pull-down with magnetic beads (GE Healthcare) was performed. Phages (5×10 10 phages) and beads were blocked with ChemiBlock. To deplete phages displaying potentially sticky scFv fragments, phages were incubated with magnetic beads coated with an irrelevant protein (biotinylated GFP). Anti-FLAG pull-down was then performed using magnetic beads coated with biotinylated anti-FLAG antibodies. After extensive washing, bound phages were eluted using FLAG peptide. After subsequent infection of XL1-Blue cells, 2.4E+06 colonies were obtained, representing clones with randomly paired feline antibody VL-lambda and VH chains.

[0220] Example 4: Expression of scFv fragments and confirmation of expression To evaluate the expression levels of the feline scFv fragments enriched during anti-FLAG pulldown, the scFv-encoding gene fragments were polyclonally cloned into the bacterial expression vector pFeBx via the restriction enzymes NcoI / EcoRI. The purified DNA fragments were ligated into the pre-cut pFeBx vector and subsequent transformation in chemically competent E. coli BL21(DE3) was performed according to standard procedures.

[0221] For analysis, 380 random clones were selected and subjected to scFv production and expression confirmation. In summary, wells of an expression plate (round-bottom 96-well plate (e.g., Thermo Scientific) Fisher, Cat. No. 262162) were filled with 120 μl / well of all-in-one medium (2x YT medium containing 34 μg / ml Cam, 0.1% glucose, and 0.5 mM IPTG) and inoculated with a single colony from the agar plate of the subcloning procedure. The plate was incubated at 37°C for 5 hours with shaking at 400 rpm and then incubated overnight at 22°C. To prepare the crude bacterial lysate used for screening purposes, the so-called BEL lysate, 40 μl / well of lysis buffer (2x BBS containing 2.5 mg / ml lysozyme, 4 mM EDTA, and 13 U / ml Benzonase) was added and incubated at 22°C for 1 hour with shaking at 400 rpm. Then 40 μl / well of blocking buffer (1x PBS containing 5% milk powder) was added and incubated at 22°C for 1 hour.

[0222] For screening, BEL lysates were captured on Maxisorp microtiter plates using coated anti-cat IgG (H+L) antibody (Jackson). Following immobilization, the plates were washed three times with PBST and then blocked with 5% milk in PBS for 1 hour at room temperature. The plates were washed three times with PBST and then anti-FLAG-HRP detection antibody (R&D Systems, HAM85291) was added to the plates and incubated for 1 hour at room temperature. The plates were washed five times with PBST and detection of bound antibodies was performed in a Tecan Genious Reader (excitation filter: 320 nm, emission filter: 430 nm) using QuantaBlu reagent according to the manufacturer's instructions.

[0223] ELISA revealed expression signals ranging from 1 to 19 times signal to background (S / BG) ​​(see FIG. 2). 62% of the clones showed S / BG expression rates of more than 10 times. The results of the ELISA expression analysis are shown in FIG. 7. The 96 best expressing scFv clones were sent for sequencing, yielding 65 unique antibody sequences. Regions representing promising building blocks for the synthetic library are shown in Tables 6 and 7, which show the feline protein sequences of framework 1 (FW1) to framework 3 (FW3) of the light and heavy chains, respectively. [Table 6] TIFF2025508603000019.tif216155 TIFF2025508603000020.tif216155 TIFF2025508603000021.tif216155 TIFF2025508603000022.tif232155 [Table 7] TIFF2025508603000024.tif232155 TIFF2025508603000025.tif216155 TIFF2025508603000026.tif216155 TIFF2025508603000027.tif200155

[0224] Example 5: Expression of selected clones in Fab format and expression confirmation All unique scFv clones identified in Example 4 were monoclonally converted to Fab format to allow soluble expression of the Fab fragments. For this purpose, each VL and VH were PCR amplified individually and stitched together via OE-PCR to introduce the constant light chain region (CL) and the VH promoter and constant region (CH1), while simultaneously removing the 18 amino acid linker previously used for the scFv format. The paired cysteine-less feline wild type and mutant constant regions are shown in Table 8. [Table 8]

[0225] Single clones were selected and Fab fragments were produced and tested essentially as described in Example 4. ELISA was performed to capture soluble Fabs using coated anti-cat IgG antibody and detection was performed on the Fab FLAG tag using anti-FLAG antibody-HRP. ELISA revealed an expression signal of up to 16-fold higher for S / BG. The results of the expression analysis by ELISA are shown in FIG. 8. Surprisingly, there was a good correlation between the expression in the scFv and Fab fragments, making these framework combinations promising scaffolds for use in phage display libraries of different formats. The correlation of the expression levels of the feline antibody fragments in scFv and Fab formats is shown in FIG. 9. The 21 highest expressing Fab clones were sent for sequencing to confirm the sequence data already obtained in the scFv format. The FW1-3 regions of the VH sequences are shown in FIG. 1 and the FW1-3 regions of the VL sequences are shown in FIG. 2.

[0226] Example 6: Construction of phage display vectors containing favorable VH / VL pairs compatible with the introduction of diversity in HCDR1, HCDR2, HCDR3, and LCDR3. For the generation of a fully synthetic feline phage display library, the most promising light and heavy chain pairs identified and verified in Examples 3-5 were synthesized as DNA fragments. Using the Geneious software suite, the sequences were codon-optimized for expression in E. coli, but rare codons were removed for expression in mammalian hosts, e.g., a triplet CGC was used to code for the amino acid arginine, since this is compatible with both bacterial and mammalian expression systems. NheI / PstI restriction sites were introduced to allow diversification of the HCDR1 / HCDR2 segments, and BssHII / XhoI were used to introduce HCDR3 diversity. For the introduction of the BssHII site, it is mandatory to use a CAR motif at the beginning of the HCDR3 region. However, this motif is the major variant in any case. BbsI / KpnI restriction sites are used to introduce LCDR3 diversity. Modifications in the heavy chain start motif to DVQL can be introduced to allow bulk reformatting between antibody formats, which can be achieved by the introduction of an MfeI site in the QL nucleotide sequence. Importantly, these modifications represent sequences that naturally occur in the feline IgG repertoire. These modified VL / VH pairs can be used for phage display in both scFv and Fab formats. The concept of library building blocks is shown in Figures 4 and 5. For the initial library, only the LCDR3 and HCDR3 regions are diversified, but candidates derived from such libraries can be easily optimized using the introduction of maturation modules in the HCDR1 and HCDR2 regions. Alternatively, it is possible to generate libraries that are diversified in all mentioned CDRs from the start.

[0227] A schematic of the building blocks for seven exemplary antibody scaffolds for the VH library is shown in FIG. 9 (SEQ ID NO: 225 is DVQLVESGGDLVKPGGSLRLTCVASGFTFSDYDMSWVRQAPGKGLQWVAAISYSGGGTGYSDSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR and SEQ ID NO: 226 is DVQLVESGGDLVKPGGSLRLTCVASGFTFSNYGMSWVRQAPGKGLQWVAAISGTGSSTYYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR). SEQ ID NO: 227 is DVQLVESGGDLVKPGGSLRLTCVASGFTFSNYDMSWVRQAPGKGLQWVAAIAYSGGNTGYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR, and SEQ ID NO: 228 is DVQLVESGGDLVKPGGSLRLTCVASGFTFSRYGMSWVRQAPGKGLQWVAAISGSGDSTYYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR. SEQ ID NO:229 is DVQLVESGGDLVQPGGSLRLTCVASGFTFSSYEMNWVRQAPGKGLQWVAYISSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR; SEQ ID NO:230 is DVQLVESGGDLVKPGGSLRLTCVASGFTFSSYYMHWVRQAPGKGLQWVAQISDSGGSTYYADSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR; SEQ ID NO:231 is DVQLVQSGGDLVK A schematic of the building blocks for seven exemplary antibody scaffolds for the VL library is shown in FIG. 10 (SEQ ID NO: 218 is QSGPNQPSSVSGALGQRVTISCTGIDTYVGWFQQIPGMAPKTIIVGNTNRPSGVPDRFSGSSKSGNTGTLTITGLQAEDEADYYC and SEQ ID NO: 219 isQSGPNQPSSVSGALGQRVTISCTGVGNYVDWYQQVPGMAPKTIIYGNRYRPSGVPDRFSGSKSGSTGTLTITGLQAEDEADYYC, SEQ ID NO: 220 is QSGPNQPSSVSGTLGQTITISCTGVSVFVDWYQQIPGMAPKTVIYRNTNRLSEVPDRFSGSKSGITATLTITGLQAEDEADYYC, SEQ ID NO: 221 is QSVVTQPPSVSGALGQRVTISCTGSSSNVGRGNYVSWYQQLSGTPPKLLIYGNTDRLSGVPDRFSGSKSGTTGSLTITGLQTEDEADYYC, SEQ ID NO: 222 is , QSGPNQPSSVSGALGQRVTISCTGAGSYVGWYQQVPGMAPKTIIYDDNKRPSGVPARFSGSKSGSTGTLTITGLQAEDEANYYC, SEQ ID NO: 223 is QPVLTQPPSVSGALGQRVTISCTGSSSNIGRGNYVSWYQQVSGTAPKLLIYGDTNRPSGVPDRFSGSKSGSTGSLTITGLQADDEADYYC, SEQ ID NO: 224 is QPVLTQPPSVSGSLGQRVTISCTGTSSNIGSGNYVSWYRQVPGIAPSLLIYENFKRPSGVPDRFSGSKSGSSGSLTITGLQADDEVDYYC).

[0228] Example 7: Design of HCDR3 diversity modules HCDR3 and LCDR3 are the main paratopes that form the CDRs in antibodies, which contribute significantly to antigen binding and recognition. Therefore, the naive library is exclusively diversified in the CDR3 region. For affinity maturation and framework diversification, our library further allows combinatorial diversification of HCDR1 and HCDR2.

[0229] Heavy chain framework 4 (C-terminal to HCDR3) is highly conserved in the feline IgG repertoire. Therefore, this region was kept constant (SEQ ID NO: 209) to facilitate molecular cloning of the HCDR3 diversity module. Furthermore, we selected a CAR motif preceding HCDR3 to allow the insertion of a BssHII restriction site.

[0230] As described in Example 1, HCDR3 lengths of 7 to 14 amino acids were used (HCDR3 starts at position 98 according to the numbering scheme shown in Table 9 and Figure 9). This selection represents 91% of all lengths identified in our NGS dataset. Furthermore, it has been shown that for therapeutic antibodies, very short or long HCDR3s are often not characterized by the required stability. [Table 9] [Table 10]

[0231] Since each length has a slightly adapted amino acid composition and differs gradually from shorter to longer HCDR3, we synthesized all lengths using the individual compositions identified in our NGS data set.The distribution of amino acids in the diversification modules of different lengths is shown in Figure 11, Figure 12, Figure 13, and the percentage of each CDR length is shown in Table 9.

[0232] Briefly, the strategy for DNA synthesis is as follows. 1) Each length is calculated in silico individually, using the individual amino acid frequencies for each position, removing cysteines and PTM motifs (e.g., glycosylation motifs, asparagine deamidation sites, aspartate isomerization sites, integrin binding sites, fragmentation sites, and hydrophobic stretches). 2) Based on the distribution of HCDR3 lengths, a fraction of the total 1E+10 sequences is preselected in silico to include the set of sequences that best represent the sequence space for each set of lengths. The total number of sequences synthesized across all lengths used is approximately 1E+10. 3) These 1E+10 sequences are synthesized using TWIST Bioscience technology to obtain an HCDR3 library that mimics the natural length and amino acid distribution found in cats.

[0233] Example 8: Design of LCDR3 LCDR3 lengths of 9 to 12 amino acids were used for the diversification modules (LCDR3 starting at position 84 according to the numbering scheme shown in Table 10 and Figure 10). This selection represents 93% of all lengths identified in our NGS dataset, as described in Example 1. Furthermore, it has been shown that for therapeutic antibodies, very short or long LCDR3s are often not characterized by the required stability. Light chain framework 4 (C-terminus of LCDR3) is conserved in the feline IgG repertoire. Therefore, this region was kept constant (SEQ ID NO: 208) to facilitate molecular cloning of the LCDR3 diversity module.

[0234] Since each length has a slightly adapted amino acid composition, and the shorter LCDR3 is gradually different from the longer LCDR3, we synthesized all lengths using their individual compositions identified in our NGS data set.The amino acid distribution in the diversification module of different lengths is shown in Figure 14, and the percentage of each CDR length is shown in Table 10.

[0235] The strategy for DNA synthesis was essentially as described for HCDR3, with similar care taken to remove cysteines and unwanted PTM motifs.

[0236] Example 9: Design of HCDR1 and HCDR2 maturation modules. To allow affinity maturation, our VH sequences feature NheI / PstI restriction sites for rapid diversification of the HCDR1-FW2-HCDR2 segment. The HCDR1-HCDR2 design is as follows: Both HCDR1 and HCDR2 are calculated in silico using the individual amino acid frequency for each position, removing cysteine ​​and PTM motifs. Since the FW2 sequence is relatively conserved for the most promising sequences identified in Examples 3-5, the FW2 sequence was kept constant (WVRQAPGKGLQWV, SEQ ID NO: 241) to improve the functionality and maturation capacity of our fully synthetic library. We selected only the two most prominent CDR lengths for HCDR1 and HCDR2, since we did not observe significant variation in length during NGS analysis.

[0237] The mature modules were synthesized using TWIST Bioscience technology to obtain an HCDR1-HCDR2 library that mimics the natural amino acid distribution found in cats. For HCDR3 and LCDR3, all unfavourable amino acid motifs were avoided.

[0238] The concept and amino acid distribution of the HCDR1 / 2 maturation module is shown in FIG. 15, which shows the adjacent region 5′ to the HCDR1 region, the diversified HCDR1, FW2, the diversified HCDR2, and the subsequent part of FW3. No length variation was implemented in this module. aa defines a random amino acid (Xaa can be any one of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). FW2 is conserved for all library members.

[0239] Example 10: Antibody Selection Phage display selection may be performed as described below or by other methods known to those skilled in the art. For example, parallel panning strategies (e.g., solution panning, Fc capture panning, direct solid-phase panning) are performed to maximize the chance of identifying a variety of binding antibodies with the desired biophysical characteristics. Various soluble proteins can be selected as model antigens (e.g., lysozyme, GFP) for library validation. Ensemble screening against three model antigens was performed on antigens immobilized on a plastic surface in direct solid-phase panning as described below. Selection against model antigens can also be performed in solution mode as described below.

[0240] Solid-phase panning For solid-phase panning, antigens were immobilized on the surface of microtiter plates (Maxisorp, 96-well flat bottom) overnight at 4°C. A coating check with each antigen was performed to identify the ideal coating concentration. Typically, antigens were used in the first panning round at a concentration of 1 μg / ml, which represents antigen saturation on the plate. For each selection, 300 μl / well of antigen diluted in PBS (phosphate-buffered saline, pH 7.4) was used. After incubation, the antigen solution was removed and the wells were washed twice with PBST (PBS supplemented with 0.05% Tween-20) before adding 400 μl / well of blocking buffer (typically 5% milk in PBS). The plates were incubated at room temperature for at least 1 hour.

[0241] In parallel, library phages were blocked with a blocking solution, typically 5% milk in PBS, for at least 1 hour at room temperature. Depending on whether the target antigen contained a tag, e.g., biotin or His tag, or was an Fc fusion protein, additional reagents were supplemented to the blocking buffer. Also, before using the phages in panning, a depletion step in relevant materials was performed (e.g., adsorption onto wells coated with unrelated biotinylated proteins to remove potential biotin-binding agents).

[0242] For each panning subcode, 20 ml of 2xYT medium was incubated with E. coli TG1F+ from M9 minimal agar plates in a phage-free working space, and the cultures were then used for infection with the selected phages. Cultures were incubated at an OD of 0.6. 600nm The E. coli cultures were kept on ice until needed for infection with the eluted phages.

[0243] After antigen coating and blocking, the blocked phages were transferred to each well and incubated for 1-2 hours at room temperature. Then, the phages were removed by quickly inverting the plate over a phage waste container. To further remove non-specific and weak phages, several washing steps with PBST and PBS were performed. In the first panning, standard washing steps were applied (3 times PBST rapidly, 2 times PBST for 5 min, 3 times PBS rapidly, 2 times PBS for 5 min). Depending on the phage product titer, the stringency was adapted in the following rounds by increasing the number and time of washing steps.

[0244] Following antigen-phage incubation and washing, specific phages were typically eluted by addition of trypsin (250 μl of a 10 μg / ml trypsin solution in PBS, 30 min at 37° C.) to cleave the proteinase-sensitive linker between the antibody fragment and the gIII protein. Alternatively, a pH shift was applied to disrupt the antigen-antibody interaction. This could be achieved using either acidic or basic buffers, but typically a low pH elution was used. For this purpose, 300 μl of elution buffer (100 mM glycine / HCl, 0.5 M NaCl, pH 2.2) was added to each well, incubated for 10 min, and then transferred to a new tube containing 10 μl of neutralization buffer (2 M Tris base).

[0245] After neutralization, the phage suspensions of each selection were transferred to pre-warmed E. coli TG1F+ cultures, respectively, and incubated at 37° C. in a water bath for 45 min without shaking. The bacterial cultures were centrifuged, the supernatants discarded, and the pellets resuspended in 2xYT medium, plated on LB / Cam agar plates, and incubated overnight at 37° C. The next day, bacteria were scraped off the plates with freezing medium (2xYT medium containing 34 μg / ml chloramphenicol (Cam), 1% glucose, and 15% glycerol), aliquots were stored at −80° C., and then phages were prepared for subsequent panning rounds.

[0246] Solution and Semi-Solution Panning For solution and semi-solution panning, magnetic beads (GE, Sera-Mag Streptavidin-Coated Magnetic Particles, Cat. No. 30152104010150) were used in combination with biotinylated model antigens to capture antigen-phage complexes or immobilize antigens on the bead surface, respectively.

[0247] Before the start of panning, the beads were washed and blocked, and for semi-solution panning, the beads were loaded with antigen. For this purpose, 250 μl of beads for each selection were transferred to a 2 ml low-binding tube, the beads were captured with a magnetic particle separator, and the storage solution was removed. The beads were then washed three times with PBS, the beads were collected using a magnet, and the washing buffer was removed. Afterwards, the beads were blocked in blocking solution (100% Chemiblock or 5% BSA in PBS) for 2 hours at room temperature.

[0248] In parallel, phages were blocked. For this purpose, the required amount of phages was mixed with an equal volume of 2x blocking solution and incubated at room temperature for at least 1 h. Typically, Chemiblock was used, supplemented with tags or irrelevant proteins containing biotin for blocking these structures. In addition, blocked phages were pre-adsorbed onto empty magnetic beads to remove sticky phages.

[0249] After blocking of the beads and blocking / pre-adsorption of the phages, biotinylated antigen was added to the phage solution and incubated at room temperature for 1 h with rotation. For capture of the phage / antigen complex, 100 μl of blocked magnetic beads were added and incubated at room temperature for 15 min. Then, non-specific phages were removed by washing (5 times PBST rapidly, 3 times PBST for 5 min, 3 times PBS rapidly), and the washing stringency was adapted for each round depending on the panning product. In the last washing step, the magnetic beads with the captured antigen-phage complex were transferred into a new low-binding tube.

[0250] For elution of specific phages, 300 μl of elution buffer was added for 10 min at room temperature. As with solid-phase panning, elution can be performed using acidic or basic conditions or by addition of trypsin. The phage suspension of each selection was then transferred to 20 ml of pre-warmed E. coli TG1F+ culture each and incubated at 37° C. in a water bath without shaking for exactly 45 min. The bacterial culture was centrifuged at 4° C. at 4600 rpm for 5 min and the supernatant was discarded. The pellet was resuspended in 600 μl of 2×YT medium and plated on a large LB / Cam agar plate and incubated overnight at 37° C. The next day, bacteria were scraped off the plates using a sterile Drygalski spatula with a few mL of freezing medium (2x YT medium containing 34 μg / ml Cam, 1% glucose, and 15% glycerol) and aliquots were stored at -80°C before preparing phages for subsequent panning rounds.

[0251] Semi-solution panning was performed similarly to solution panning, with the following modifications: instead of capturing the complex of phage bound to biotinylated target from solution using streptavidin magnetic beads, the respective antigen was already immobilized on the beads before blocking of the beads. Thus, the panning mode reflects the selection on a solid phase, but allows better target orientation and washing conditions compared to panning where the antigen is coated on the surface of a microtiter plate.

[0252] The remaining steps of the panning, including washing and elution steps, are carried out essentially as described above.

[0253] Phage preparation For each phage preparation, seeding medium (2x YT medium containing 34 µg / ml Cam and 1% glucose) was inoculated with the phagemid-containing bacterial suspension or glycerol stock to an OD of approximately 0.2–0.3. 600 The cultures were incubated at an OD of approximately 0.5-0.6. 600The cells were incubated at 37°C for 30-90 min with shaking until a 10% phages concentration was reached. VCSM13 helper phage was added (4.00E+10tu helper phage was used per 5ml bacterial culture) and incubated at 37°C for 30 min without shaking, then incubated at 37°C for 30 min with shaking at 250 rpm. Afterwards, the bacteria were spun down and the helper phage containing supernatant was discarded. The phage-infected bacteria were resuspended in 2xYT medium containing induction medium (34μg / ml Cam, 50mg / ml kanamycin (Kan and 0.2mM IPTG) and incubated at 22°C for 18-20 h with shaking at 200 rpm in a phage shaker. The next day, the bacteria were spun down and the supernatant containing the antibody-displayed phage was transferred to a new tube. For phage precipitation, 1 / 5 volume of ice-cold PEG / NaCl was added to the phage-containing supernatant, mixed and gently incubated at 37°C for 30 min with shaking at 250 rpm. The mixture was incubated on ice for at least 30 minutes with gentle shaking. The precipitated phages were spun down at 12000×g at 4° C. for at least 30 minutes. The supernatant was quantitatively removed and the phage pellet was resuspended in an appropriate volume of PBS. The phage titer was determined using spectrophotometer measurement (Nanodrop; absorbance at 268 nm was used as a measure for phage quantity; an absorbance of A268 nm of 1 corresponds to a phage titer of 5.00E+12 tu / ml).

[0254] Subcloning into an expression vector After multiple rounds of panning, the polyclonal phage products were subcloned into the bacterial expression vector pFeBx, which is compatible with the expression of either single chains or Fab fragments. The scFv- or Fab-encoding fragments were removed from the phage display vector using the restriction enzymes NcoI / EcoRI and NcoI / XhoI, respectively, and isolated using preparative agarose gel electrophoresis (1.2% agarose), and the fragments were DNA purified from the gel slices using an appropriate gel extraction kit. The ligation reaction between the inserts and the pre-cut pFeBx vector, as well as the subsequent transformation in chemically competent E. coli TG1F-, was carried out according to standard procedures.

[0255] The wells of an expression plate (round-bottom 96-well plate (e.g., Thermo Fisher, Cat. No. 262162) were filled with 120 μl / well of all-in-one medium (2x YT medium containing 34 μg / ml Cam, 0.1% glucose, and 0.5 mM IPTG) and inoculated with a single colony from the agar plate of the subcloning procedure. The plate was incubated at 37° C. for 5 hours with shaking at 400 rpm, and then incubated overnight at 22° C. To prepare the crude bacterial lysate used for screening purposes, the so-called BEL lysate, 40 μl / well of lysis buffer (2x BBS containing 2.5 mg / ml lysozyme, 4 mM EDTA, and 13 U / ml Benzonase) was added and incubated at 22° C. for 1 hour with shaking at 400 rpm. Then, 40 μl / well of blocking buffer (1x PBS containing 5% milk powder) was added and incubated at 22° C. for 1 hour. The plate was either stored at −20° C. or used directly for screening.

[0256] Screening ELISA For screening, lysates containing scFv or Fab (BEL lysates) were tested for binding to antigen immobilized on Maxisorp microtiter plates or, alternatively, using biotinylated antigen bound to streptavidin plates. Following immobilization of antigen on the corresponding surface, the plates were washed three times with PBST and then blocked with 5% milk in PBS for 1 h at room temperature. BEL lysates, control antibodies, and negative controls were transferred to the plates and incubated for 1 h at room temperature. The plates were washed three times with PBST and then detection antibodies were added to the plates and incubated for 1 h at room temperature. For detection of scFv, anti-FLAG-HRP antibody (R&D Systems, HAM85291) was used and Fab was detected with goat anti-cat IgG (L+H)-HRP (Jackson). Plates were washed five times with PBST and detection of bound antibodies was performed in a Tecan Genious Reader (excitation filter: 320 nm, emission filter: 430 nm) using QuantaBlu reagent according to the manufacturer's instructions.

[0257] Example 11. Results from a panning campaign on a model antigen eGFP (enhanced green fluorescent protein), fAG1 (feline soluble cytokine), and fAG2 (ECD of feline receptor) were selected as model antigens for library validation. Screening and sequencing data obtained from solid-phase panning and direct coating ELISA above are presented, demonstrating that antigen-specific binders from a fully synthetic feline phage library can be isolated.

[0258] Antigen binding was tested using scFv-containing BEL lysate. Primary hits were defined as antibody fragments that produced ELISA signals at least 60 times higher than background, and their specificity to model antigen was confirmed in secondary ELISA in unrelated control antigen. Exemplary screening data for eGFP after solid-phase panning is shown in dot blot visualization in Figure 16, which shows strong target-specific binding and only minimal binding to unrelated proteins.

[0259] A total of 312, 139, and 24 hits were identified for eGFP, fAG1, and fAG2, respectively, for all three test pannings, representing overall hit rates ranging from 13% to nearly 90% (see Table 11). [Table 11]

[0260] The heavy and light chain CDR3 regions of promising clones for each panning product were sequenced to estimate sequence diversity. As shown in Table 11, sequence diversity was high. For eGFP, all sequenced antibody sequences were unique. The remaining test panning products also contained a few sequences enriched during the panning process, and therefore the overall sequence diversity was slightly lower (approximately 30% for fAG1 and 60% for fAG2). As expected, the sequences covered a variety of CDR lengths in both HCDR3 and LCDR3 (data not shown).

[0261] Further exemplary embodiments of the present invention include the following. 1. A polypeptide library comprising a VH polypeptide member comprising a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, wherein the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment is selected from the group consisting of: (D / Q / Y / E)(V / E)(Q / L / R)LV(E / Q)SGGD(L / R)V(K / Q)PGGGSLRL(T / I / A)C(V / M)(A / G)SGF(T / P / I / N)(F / V / L)XaaXaaXaaXaaXaaXaaXaaWVRQ(A / T)PG(K / M)GLQWVXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa aXaaDSVKGRFT(I / V)S(R / K)D(N / D)(A / P / V)(K / R / M / E)NTL(Y / L)LQM(N / T / D)(S / G / N)LKTED(T / A / M)ATYYC(A / S / T)(R / K / N / T / G)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaWG(Q / R)G(A / T / V)(L / P)VTVSS (SEQ ID NO: 232); Preferably, DEQLVESGGDLVKPGGSLRLTCVASGFPF (SEQ ID NO: 240), DEQLVESGGDLVKPGGSLRLTCVASGFTL (SEQ ID NO: 241), DEQLVESGGDLVKPGGSLRLTCVGSGFTF (SEQ ID NO: 242), DVQLVESGGDLVKPGGSLRLACVASGFTF (SEQ ID NO: 243), DVQLVESGGDLVKPGGSLRLICVASGFTF (SEQ ID NO: 244), DVQLVESGGDLVKPGGSLRLTCVASGFIF (SEQ ID NO: 245), DVQLVESGGDLVKPGGSLRLTCVASGFPF (SEQ ID NO: 246), DVQLVESGG and / or comprising a FW1 sequence selected from DLVKPGGSLRLTCVASGFTF (SEQ ID NO: 247), DVQVVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 248), DVRLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 249), DVRLVESGGDRVKPGGSLRLTCVASGFTF (SEQ ID NO: 250), EVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 251), QVLLVQSGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 252), or YVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 253); and / or Preferably, it comprises an HCDR1 sequence selected from GSYDMT (SEQ ID NO: 254), NNFAMS (SEQ ID NO: 255), NSYAMS (SEQ ID NO: 256), RGYAMT (SEQ ID NO: 257), RSHWMN (SEQ ID NO: 258), SDYDMS (SEQ ID NO: 259), SGYSMN (SEQ ID NO: 260), SLYDMS (SEQ ID NO: 261), SNYDMS (SEQ ID NO: 262), SNYGMD (SEQ ID NO: 263, SNYGMS (SEQ ID NO: 264), SRYGMS (SEQ ID NO: 265, SSYAMS (SEQ ID NO: 266), SSYEMN (SEQ ID NO: 267), SSYGMS (SEQ ID NO: 268), SSYYMH (SEQ ID NO: 269), TGDAMS (SEQ ID NO: 270), or TNYAMS (SEQ ID NO: 271); and / or Preferably, it comprises a FW2 sequence selected from WVRQAPGKGLQWV (SEQ ID NO: 271), WVRQAPGMGLQWV (SEQ ID NO: 273), WVRQAPGRGLQWV (SEQ ID NO: 274), or WVRQTPGKGLQWV (SEQ ID NO: 275), and / or Preferably, AAIAYNGGNTGYA (SEQ ID NO: 276), AAIGHDGSTTAYA (SEQ ID NO: 277), AAIRGSGGVTYYA (SEQ ID NO: 278), AAISGSGDSTYYA (SEQ ID NO: 279), AAISYNGGGTGYS (SEQ ID NO: 280), AANSGTGSSTYYA (SEQ ID NO: 281), ADISGSGGATAYA (SEQ ID NO: 282), AGISGSGITTYYA (SEQ ID NO: 283), AGISTSGGNTYYA (SEQ ID NO: 284), AGITSGGNTYYA (SEQ ID NO: 285), AQISDSGGSTYYA (SEQ ID NO: 286), AYIDNDGSS and / or comprising an HCDR2 sequence selected from TYYA (SEQ ID NO: 287), AYIRYDGNTIHYG (SEQ ID NO: 288), AYIRYDGSSTNYA (SEQ ID NO: 289), AYISSGGSTYYA (SEQ ID NO: 290), CAIGGTGSRTLYA (SEQ ID NO: 291), SAISFDGSGTGYA (SEQ ID NO: 292), SALSESGHSTIYA (SEQ ID NO: 293), SSISSGGTTYYA (SEQ ID NO: 294), STIDSGGNTHYI (SEQ ID NO: 295), TDISRSGATTYYA (SEQ ID NO: 296), or TTISGSGGSTYYA (SEQ ID NO: 297); Preferably, DSVKGRFTISKDDAENTLYLQMNSLKTEDTATYYCAG (SEQ ID NO: 298), DSVKGRFTISRDNAENTLLLQMNSLKTEDTATYYCAR (SEQ ID NO: 299), DSVKGRFTISRDNAKNTLSLQMDSLKTEDTATYYCAT (SEQ ID NO: 300), DSVKGRFTISRDNAKNTLYLQMDSLKTEDTATYYCTS (SEQ ID NO: 301), DSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR (SEQ ID NO: 302), DSVKGRFTISRDNAKNTLYLQMNNLKTEDTATYYCAG (SEQ ID NO: 303), DSVKGRFTISRDNAKNTLYLQMNSLKTEDMATYYCAR (SEQ ID NO: 304), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTAT and / or comprising a FW3 sequence selected from YYCAK (SEQ ID NO: 305), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 306), DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAT (SEQ ID NO: 307), DSVKGRFTISRDNAMNTLYLQMNSLKTEDAATYYCAR (SEQ ID NO: 308), DSVKGRFTISRDNARNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 309), DSVKGRFTISRDNPKNTLYLQMTSLKTEDTATYYCAR (SEQ ID NO: 310), DSVKGRFTISRDNVKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 311), or DSVKGRFTVSRDNAKNTLYLQMNSLKTEDTATYYCSR (SEQ ID NO: 312); and / or Preferably, the FW4 sequence is selected from WGQGALVTVSS (SEQ ID NO: 213), WGQGAPVTVSS" (SEQ ID NO: 214), WGQGTLVTVSS (SEQ ID NO: 215), WGQGVLVTVSS (SEQ ID NO: 216), and WGRGALVTVSS (SEQ ID NO: 217), and / or Optionally, VH FW1 is DVQLVESGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 247), DVQLVESGGDLVQPGGSLRLTCVASGFTF (SEQ ID NO: 405), and / or DVQLVQSGGDLVKPGGSLRLTCVASGFTF (SEQ ID NO: 406), and / or Optionally, the HCDR1 sequence is SDYDMS (SEQ ID NO: 259), SNYGMS (SEQ ID NO: 264), SNYDMS (SEQ ID NO: 262), SRYGMS (SEQ ID NO: 265), SSYEMN (SEQ ID NO: 267), SSYYMH (SEQ ID NO: 269), and / or SSYAMS (SEQ ID NO: 266); and / or Optionally, the VH FW2 sequence is WVRQAPGKGLQWV (SEQ ID NO: 271), and / or Optionally, the HCDR2 sequence is AAISYNGGGTGYS (SEQ ID NO: 280), AANSGTGSSTYYA (SEQ ID NO: 281), AAIAYNGGNTGYA (SEQ ID NO: 276), AAISGDSTYYA (SEQ ID NO: 279), AYISSGGSTYYA (SEQ ID NO: 290), AQISDSGGSTYYA (SEQ ID NO: 286), and / or ADISGSGGATAYA (SEQ ID NO: 282); and / or Optionally, the VH FW3 sequence is DSVKGRFTISRDNAKNTLYLQMNGLKTEDTATYYCAR (SEQ ID NO: 302), or DSVKGRFTISRDNAKNTLYLQMNSLKTEDTATYYCAR (SEQ ID NO: 306), and / or Optionally, the VH FW4 sequence is WGQGALVTVSS (SEQ ID NO: 213), or alternatively, the VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segments comprise substantially identical sequences.

[0262] 2. A VL polypeptide member comprising a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, wherein the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment is Q(S / P / E)(V / G)(P / L / V)(T / N)Q(P / E)(P / S / T)S(V / L / M)S(G / A / T)(A / G / S / T)(L / P)G(Q / G / T)(R / A / T / K)(V / I)(T / R)(I / M / L)(S / T)CXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaW(Y / V)(Q / R)Q(I / V / L / K / H)(P / S)(G / R / Y)(M / K / R / Q / T / S)(A / P / R)(P / F)(K / Q / S)(T / L / A / V / Y)(L / I / V / F)(I / V / L)(Y / V / G / H)(- / Y)(- / Y)(- / S)(- / D)XaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(D / A / N / S)R(F / I / L / V)SGS(K / L)(- / D)(- / A)(S / V)(G / A)(S / N / T / Q / I / A)(T / K / A)(G / A)(S / T / L / V)L(T / A / L)(I / V)(T / S)G(L / P / A)Q(A / S / T / P)EDE(A / G / V)(D / N)Y(Y / H)CXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa(F / I)G(G / R)GTH(L / V)(T / S)VL(SEQ ID NO: 236) and comprising at least one sequence selected from the group consisting of: Preferably, QSVVTQPPSVSGALGQAVTISC (SEQ ID NO: 313), QSVLTQPPSVSGALGQTVTISC (SEQ ID NO: 314), QSVLTQPPSVSGGLGQKVTISC (SEQ ID NO: 315), QPVVTQPPSVSGALGQTVTISC (SEQ ID NO: 316), QSVLTQPPSMSGALGQTVTISC (SEQ ID NO: 317), QPVLTQPPSVSGSLGQRVTISC (SEQ ID NO: 318), QSGPNQPSSVSGALGQRVTISC (SEQ ID NO: 319), QSGPNQPSSVSGALGQRVTMSC (SEQ ID NO: 320), QSGPNQPSS and / or comprising a FW1 sequence selected from VSAALGQRVTISC (SEQ ID NO: 321), QSGPNQPSSVSGTLGQTITISC (SEQ ID NO: 322), QEVVTQETSLSTTPGGTVTLTC (SEQ ID NO: 323), QSVVTQPPSVSGALGQRVTISC (SEQ ID NO: 324), QPVLTQPPSVSGALGQRVTISC (SEQ ID NO: 325), QPVVTQPPSVSGALGQRVTISC (SEQ ID NO: 326), QPVLTQPSSLSASPGTTARLTC (SEQ ID NO: 327), or QPVVTQPPSLSGSLGATARLTC (SEQ ID NO: 328); and / or Preferably, AGSANNIGITDVN (SEQ ID NO: 329), AGSGSNIGIVDVN (SEQ ID NO: 330), AGSGSNIGIAGVN (SEQ ID NO: 331), AGSANNNGIVGVN (SEQ ID NO: 332), AGSGSNIGIVAGVN (SEQ ID NO: 333), TGTSSNIGSGNYVS (SEQ ID NO: 334), TGIDTYVG (SEQ ID NO: 335), TGVDTYVD (SEQ ID NO: 336), TGVDSYVG (SEQ ID NO: 337), SGAGSYVA (SEQ ID NO: 338), TGAGSYVG (SEQ ID NO: 339), TGV and / or comprising an LCDR1 sequence selected from GNYVD (SEQ ID NO: 340), TGVGMSVD (SEQ ID NO: 341), TGAGYVG (SEQ ID NO: 342), TVGSVFVD (SEQ ID NO: 343), GSSTGAVTTSNFAS (SEQ ID NO: 344), TGSSSNVGRGNYVS (SEQ ID NO: 345), TGSSPNIGRGNYVS (SEQ ID NO: 346), TGSSSNIGRGNYVS (SEQ ID NO: 347), TLSSGFNVGGYYIS (SEQ ID NO: 348), or TLSREVSVGVKGIY (SEQ ID NO: 349); and / or Preferably, WYQQLPGKAPKLLIY (SEQ ID NO: 350), WYQQLPGKAPSLLIY (SEQ ID NO: 351), WYQQHPRKAPKLLIY (SEQ ID NO: 352), WYQQLPGKAPQLLIY (SEQ ID NO: 353), WYQQLPGKAPKALIY (SEQ ID NO: 354), WYRQVPGIAPSLLIY (SEQ ID NO: 355), WFQQIPGMAPKTIIV (SEQ ID NO: 356), WYQQIPGMAPKTIIY (SEQ ID NO: 357), WYQQIPGRAPKTIIY (SEQ ID NO: 358), WYQQVPGMAPKTI and / or comprising a FW2 sequence selected from: IY (SEQ ID NO: 359), WYQQIPGMAPKTVIY (SEQ ID NO: 360), WVQQKPYQRFQGLVG (SEQ ID NO: 361), WYQQLSGTPPKLLIY (SEQ ID NO: 362), WYQQLSGTAPKVLIY (SEQ ID NO: 363), WYQQLSGTAPKLLIY (SEQ ID NO: 364), WYQQVSGTAPKLLIY (SEQ ID NO: 365), WFQQKPGSPPRYLLYYYSD (SEQ ID NO: 366), or WYQQKPGSPPRYFLHYYSD (SEQ ID NO: 367); and / or Preferably, ANNRRPSSVP (SEQ ID NO: 368), ASDRRPSGVP (SEQ ID NO: 369), GSSNRPSGVP (SEQ ID NO: 370), ASTRRPSGVP (SEQ ID NO: 371), ENFKRPSGVP (SEQ ID NO: 372), GNTNRPSGVP (SEQ ID NO: 373), YDSNRPSGVP (SEQ ID NO: 374), DNSNRPSGVP (SEQ ID NO: 375), DDNKRPSGVP (SEQ ID NO: 376), GNRYRPSGVP (SEQ ID NO: 377), and / or comprises an LCDR2 sequence selected from RNSNRLSEVP (SEQ ID NO: 378), GNSYRPSGVP (SEQ ID NO: 379), RNTNRLSEVP (SEQ ID NO: 380), GTSYRNPGVP (SEQ ID NO: 381), GNSDRLSGVP (SEQ ID NO: 382), GDKNRPSGVP (SEQ ID NO: 383), GDTNRPSGVP (SEQ ID NO: 384), SDKHQGPGVP (SEQ ID NO: 385), or STNELGPGVP (SEQ ID NO: 386); and / or Preferably, DRFSGSKSGATGSLTISGLQAEDEADYYC (SEQ ID NO: 387), ERISGSRSGNTGSLTITGLQTEDEADYYC (SEQ ID NO: 388), DRFSGSKSGNTGSLTITGLQAEDEADYYC (SEQ ID NO: 389), DRFSGSKSGNTGSLTITGLQAEDEADYYC (SEQ ID NO: 390), ERLSGSKSGNTGSLTITGLQPEDEADYYC (SEQ ID NO: 391), DRFSGSKSGSSGSLTITGLQADDEVDYYC (SEQ ID NO: 392), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 393), DRFSGSKSGSTGTLAITGLQAEDEGDYYC (SEQ ID NO: 394), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO: 395), DRFSGSKSG and / or comprising a FW3 sequence selected from NTGTLTITGLQAEDEADYYC (SEQ ID NO: 396), ARFSGSKSGSTGTLTITGLQAEDEANYYC (SEQ ID NO: 397), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 398), NRFSGSKSGSTATLTITGLQAEDEADYYC (SEQ ID NO: 399), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO: 400), DRFSGSKSGITATLTITGLQAEDEADYYC (SEQ ID NO: 401), ARFSGSLVGQKAVLTITGAQSEDEAEYYC (SEQ ID NO: 402), DRFSGSKSGTTGSLTITGLQTEDEADYYC (SEQ ID NO: 403), or DRFSGSKSGSTGSLTITGLQAEDEADYYC (SEQ ID NO: 404); and / or Preferably, it comprises a FW4 sequence selected from FGGGTHLTVL (SEQ ID NO: 208), FGGGTHLSVL (SEQ ID NO: 209), FGRGTHLTVL (SEQ ID NO: 210), FGGGTHVTVL (SEQ ID NO: 211) or IGGGTHVTVL (SEQ ID NO: 212), and / or Optionally, the FW1 sequence is QPVLTQPPSVSGSLGQRVTISC (SEQ ID NO: 318), QSGPNQPSSVSGALGQRVTISC (SEQ ID NO: 319), QSGPNQPSSVSGTLGQTITISC (SEQ ID NO: 322), QSVVTQPPSVSGALGQRVTISC (SEQ ID NO: 324), and / or QPVLTQPPSVSGALGQRVTISC (SEQ ID NO: 325). Optionally, the HCDR1 sequence is TGTSSNIGSGNYVS (SEQ ID NO: 334), TGIDTYVG (SEQ ID NO: 335), TGAGSYVG (SEQ ID NO: 339), TGVGNYVD (SEQ ID NO: 340), TVGSVFVD (SEQ ID NO: 343), TGSSSNVGRGNYVS (SEQ ID NO: 345), and / or TGSSSNIGRGNYVS (SEQ ID NO: 347). Optionally, the VL FW2 sequence is WYRQVPGIAPSLLIY (SEQ ID NO:355), WFQQIPGMAPKTIIV (SEQ ID NO:356), WYQQVPGMAPKTIIY (SEQ ID NO:359), WYQQIPGMAPKTVIY (SEQ ID NO:360), WYQQLSGTPPKLLIY (SEQ ID NO:362), and / or WYQQVSGTAPKLLIY (SEQ ID NO:365). Optionally, the LCDR2 sequence is ENFKRPSGVP (SEQ ID NO: 372), GNTNRPSGVP (SEQ ID NO: 373), DDNKRPSGVP (SEQ ID NO: 376), GNRYRPSGVP (SEQ ID NO: 377), RNTNRLSEVP (SEQ ID NO: 380), GNTDRLSGVP (SEQ ID NO: 410), and / or GDTNRPSGVP (SEQ ID NO: 384). Optionally, the VL FW3 sequence is DRFSGSKSGSSGSLTITGLQAEDEVDYYC (SEQ ID NO:411), DRFSGSKSGNTGTLTITGLQAEDEADYYC (SEQ ID NO:396), ARFSGSKSGSTGTLTITGLQAEDEANYYC (SEQ ID NO:397), DRFSGSKSGSTGTLTITGLQAEDEADYYC (SEQ ID NO:400), DRFSGSKSGITATLTITGLQAEDEADYYC (SEQ ID NO:401), DRFSGSKSGTTGSLTITGLQTEDEADYYC (SEQ ID NO:403), and / or DRFSGSKSGSTGSLTITGLQAEDEADYYC (SEQ ID NO:404); Optionally, the VL FW4 sequence is FGGGTHLTVL (SEQ ID NO: 208) or alternatively, the VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segments comprise substantially identical sequences, preferably a polypeptide library as described in embodiment 1.

[0263] 3. A VL polypeptide member comprising a VL FW1-CDR1-FW2-CDR2-FW3 segment, wherein the VL FW1-CDR1-FW2-CDR2-FW3 segment is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, and / or SEQ ID NO: 224, and preferably comprising at least one sequence selected from SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 92, 3. A polypeptide library, preferably according to embodiment 1 or 2, comprising or comprising substantially identical to at least one sequence selected from SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224, and / or SEQ ID NO:224.

[0264] 4. VH comprises a VH polypeptide member comprising a FW1-CDR1-FW2-CDR2-FW3 segment,The FW1-CDR1-FW2-CDR2-FW3 segment is represented by SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, 164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:1 94, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231, preferably SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188 7, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231,a VH polypeptide member comprising a FW1-CDR1-FW2-CDR2-FW3 segment,The FW1-CDR1-FW2-CDR2-FW3 segment is represented by SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, 164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:1 94, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231, preferably SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188 7. A polypeptide library comprising, or comprising substantially identical to, at least one sequence selected from SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, and / or SEQ ID NO:231, preferably a polypeptide library according to any one of the preceding embodiments.

[0265] 5. At least one VL / VH polypeptide member combination, comprising at least one VL / VH polypeptide member combination selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:77 and SEQ ID NO:142, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:79 and SEQ ID NO:144, SEQ ID NO:80 and SEQ ID NO:145, SEQ ID NO:81 and SEQ ID NO:146, SEQ ID NO:82 and SEQ ID NO:147, SEQ ID NO:83 and SEQ ID NO:148, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:87 and SEQ ID NO:152, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:89 and SEQ ID NO:154, SEQ ID NO:90 and SEQ ID NO:155, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:93 and SEQ ID NO:158, SEQ ID NO:94 and SEQ ID NO:159, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:96 and SEQ ID NO:161, SEQ ID NO:97 and SEQ ID NO:162, SEQ ID NO:98 and SEQ ID NO:163, SEQ ID NO:99 and SEQ ID NO:164 , SEQ ID NO:100 and SEQ ID NO:165, SEQ ID NO:101 and SEQ ID NO:166, SEQ ID NO:102 and SEQ ID NO:167, SEQ ID NO:103 and SEQ ID NO:168, SEQ ID NO:104 and SEQ ID NO:169, SEQ ID NO:105 and SEQ ID NO:170, SEQ ID NO:106 and SEQ ID NO:171, SEQ ID NO:107 and SEQ ID NO:172, SEQ ID NO:108 and SEQ ID NO:173, SEQ ID NO:109 and SEQ ID NO:174, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:111 and SEQ ID NO:176, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:113 and SEQ ID NO:178, SEQ ID NO:114 and and SEQ ID NO:179, SEQ ID NO:115 and SEQ ID NO:180, SEQ ID NO:116 and SEQ ID NO:181, SEQ ID NO:117 and SEQ ID NO:182, SEQ ID NO:118 and SEQ ID NO:183, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:120 and SEQ ID NO:185, SEQ ID NO:121 and SEQ ID NO:186, SEQ ID NO:122 and SEQ ID NO:187, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:124 and SEQ ID NO:189, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:126 and SEQ ID NO:191, SEQ ID NO:127 and SEQ ID NO:192, SEQ ID NO:128 and SEQ ID NO:193,SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:130 and SEQ ID NO:195, SEQ ID NO:131 and SEQ ID NO:196, SEQ ID NO:132 and SEQ ID NO:197, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:136 and SEQ ID NO:201, SEQ ID NO:137 and SEQ ID NO:202, SEQ ID NO:218 and SEQ ID NO:225, SEQ ID NO:219 and SEQ ID NO:226, SEQ ID NO:220 and SEQ ID NO:227, SEQ ID NO:221 and SEQ ID NO:228, SEQ ID NO:222 and SEQ ID NO:2 29, SEQ ID NO:223 and SEQ ID NO:230, and / or SEQ ID NO:224 and SEQ ID NO:230, preferably comprising at least one VL / VH polypeptide member combination selected from SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:87 and SEQ ID NO:152, SEQ ID NO:89 and SEQ ID NO:160, SEQ ID NO:88 and SEQ ID NO:161, SEQ ID NO:89 and SEQ ID NO:162, SEQ ID NO:89 and SEQ ID NO:163, SEQ ID NO:89 and SEQ ID NO:164, SEQ ID NO:89 and SEQ ID NO:165, SEQ ID NO:89 and SEQ ID NO:166, 6 and SEQ ID NO:151, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:112 and SEQ ID NO:177, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, and / or A polypeptide library, preferably according to any one of the preceding embodiments, comprising a combination of sequences selected from SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:218 and SEQ ID NO:225, SEQ ID NO:219 and SEQ ID NO:226, SEQ ID NO:220 and SEQ ID NO:227, SEQ ID NO:221 and SEQ ID NO:228, SEQ ID NO:222 and SEQ ID NO:229, SEQ ID NO:223 and SEQ ID NO:230, and / or SEQ ID NO:224 and SEQ ID NO:230, or comprising a combination of substantially identical sequences.

[0266] 6. The VL polypeptide member according to embodiment 0, comprising at least one VL / VH polypeptide member combination, wherein at least one VL / VH polypeptide member combination is preferably selected from SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, and / or SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:224, and / or SEQ ID NO:224, and preferably 138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:184, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230 and / or SEQ ID NO:231, or comprising a combination of substantially identical sequences.

[0267] 7. A polypeptide library comprising at least one VL / VH polypeptide member combination, wherein the at least one VL / VH polypeptide member combination comprises at least one VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment described in embodiment 1, and a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment described in embodiment 2.

[0268] 8. A polypeptide library comprising at least one scFv or Fab antibody fragment, wherein at least one scFv or Fab antibody fragment comprises any of a VH FW1-CDR1-FW2-CDR2-FW3 segment, a VL FW1-CDR1-FW2-CDR2-FW3 segment, a VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4 segment, a VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4 segment, or a VL / VH polypeptide member combination according to any one of embodiments 1-7.

[0269] 9. The polypeptide library of embodiment 0, comprising at least one scFv fragment, wherein the VH polypeptide member is N-terminally linked to the VL polypeptide member by a linker of about 15 to about 25, preferably about 18, amino acids, preferably a linker comprising the amino acid sequence GGSSRSSSSGGGGSGGGG.

[0270] 10. The polypeptide library of any one of the preceding embodiments, wherein at least one VH polypeptide comprises a FW4 region sequence selected from the amino acid sequence WGQGAPVTVSS (SEQ ID NO: 214), WGQGTLVTVSS (SEQ ID NO: 215), WGQGVLVTVSS (SEQ ID NO: 216), and WGRGALVTVSS (SEQ ID NO: 217); preferably, the FW4 region sequence is WGQGALVTVSS (SEQ ID NO: 213).

[0271] 11. The polypeptide library of any one of the preceding embodiments, wherein at least one VL polypeptide comprises a FW4 region sequence selected from the amino acid sequence FGGGTHLTVL (SEQ ID NO:208), FGGGTHLSVL (SEQ ID NO:209), FGRGTHLTVL (SEQ ID NO:210), FGGGTHVTVL (SEQ ID NO:211), and IGGGTHVTVL (SEQ ID NO:212), and preferably one VL polypeptide comprises a FW4 region sequence selected from the amino acid sequence FGGGTHLTVL (SEQ ID NO:208).

[0272] 12. The polypeptide library of any one of the preceding embodiments, wherein the polypeptide library comprises different polypeptide members that comprise different feline-specific CDR3 polypeptide sequences between the FW3 and FW4 segments.

[0273] 13. The polypeptide library of embodiment 12, wherein the feline-specific CDR3 polypeptide sequence is a randomized or partially randomized amino acid sequence.

[0274] 14. The polypeptide library according to embodiment 13, wherein the feline-specific CDR3 polypeptide sequences are characterized by the design shown in Table 10, and preferably, the LCDR3 having a length of 7-9 amino acids has the amino acid distribution shown in Figure 14, and / or the HCDR3 having a length of 10-12 amino acids has the amino acid distribution shown in Table 9, and / or the HCDR3 having a length of 7-9 amino acids has the amino acid distribution shown in Figure 11, the HCDR3 having a length of 10-12 amino acids has the amino acid distribution shown in Figure 12, and the HCDR3 having a length of 13-14 amino acids has the amino acid distribution shown in Figure 13.

[0275] 15. The polypeptide library of any one of the preceding embodiments, wherein the VH CDR1-FW2-CDR2 sequence contained within the VH polypeptide member of any one of the preceding embodiments is replaced by an HCDR1 / 2 maturation module, preferably wherein the VH maturation module has the design shown in Figure 15.

[0276] 16. A polypeptide library according to embodiments 8 to 15, in which the scFv antibody fragment comprises an N- or C-terminal affinity tag, preferably a FLAG tag.

[0277] 17. A polypeptide library according to any one of embodiments 6 to 16, wherein at least one VL / VH polypeptide member combination comprises a full-length or truncated phage pIII polypeptide sequence linked to a VH polypeptide.

[0278] 18. The polypeptide library of any one of the preceding embodiments, wherein the polypeptide members lack post-translational modification (PTM) sites.

[0279] 19. Substantially all of the VL / VH polypeptide member combinations are a) has a monomeric content of at least 85% when expressed in E. coli in a Fab format; and / or b) has a monomeric content of at least 90% when expressed in an IgG format in a mammalian system; and / or c) A polypeptide library according to any one of embodiments 6 to 18, characterized by a Tm of 60°C or higher.

[0280] 20. The polypeptide library of any one of the preceding embodiments, wherein the library is an antibody library, preferably a feline antibody library.

[0281] 21. The polypeptide library of any one of the preceding embodiments, wherein the library is a fully synthetic or semi-synthetic antibody library.

[0282] 22. A polypeptide library according to any one of the preceding embodiments, wherein the polypeptides in the library are feline antibodies selected from single chain Fv (scFv) fragments, Fab fragments, and F(ab)2 fragments.

[0283] 23. A feline antibody comprising a VL polypeptide member, and / or a VH polypeptide member, or a VL / VH polypeptide member combination, according to any one of the preceding embodiments, wherein preferably the antibody is a complete antibody, a single chain Fv (scFv) fragment, a Fab fragment, or a F(ab)2 fragment.

[0284] 24. An antibody obtained from a polypeptide library described in any one of embodiments 1 to 22.

[0285] 25. A collection of nucleic acid molecules encoding the polypeptide library members of any one of the preceding embodiments.

[0286] 26. A collection of vectors comprising the nucleic acid molecule of embodiment 25.

[0287] 27. A recombinant host cell comprising the nucleic acid molecule of embodiment 25 or the vector of embodiment 26.

[0288] 28. A method for generating a polypeptide library, comprising: (i) providing feline cDNA generated from antibody-expressing tissue, preferably splenocytes or lymphocytes; (ii) amplifying the VL and VH sequences by using at least one forward primer, preferably selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, or substantially identical sequences, and at least one reverse primer selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or substantially identical sequences, to obtain an amplicon; (iii) generating VL / VH combination sequences by randomly ligating the amplicons obtained in step ii), (iv) optionally cloning the VL / VH combination sequence into a vector, preferably selected from a plasmid, a phagemid, or an expression module. (v) optionally expressing in a phage expression system a single chain Fv fragment comprising a VL / VH combination sequence linked to the full length of the truncated phage pIII polypeptide; (vi) optionally isolating phage expressing the VL / VH combination sequence by affinity tag, and optionally determining the sequence of the VL / VH combination sequence in the isolated phage; (vii) optionally infecting a bacterium with the phage isolated in step (vi), preferably the bacterium is E. coli; (viii) optionally subcloning the VL / VH combination sequence determined by sequencing in step (vi) into an expression system and expressing the VL / VH combination sequence as a soluble scFv fragment, preferably wherein the expression system is a bacterial expression system; (ix) optionally determining the expression level of soluble scFv fragments in the expression system of step (viii) by ELISA; (x) optionally sequencing DNA encoding VL / VH combination sequences from high expressing clones of the expression system; (xi) optionally cloning the VL and VH combined sequence determined by sequencing in step (x) into a Fab expression system; (xii) optionally expressing the Fab fragment in the cell; (xiii) optionally determining the expression level of soluble Fab fragments in the cells in step (xii); (xiv) optionally determining the sequences of the VL and VH combined sequences in the high expressing cells by sequencing; (xv) Optionally, the method comprises a step in which highly expressed VL / VH combination sequences in scFv and Fab formats are determined by comparing the sequences obtained in step (x) and step (xiv).

[0289] 29. The primers used in step (ii) are designed to incorporate a secondary primer binding site at the 5' end of the amplified VL amplicon and at the 3' end of the amplified VH amplicon, and / or to incorporate a flexible linker at the 3' end of the VL amplicon and at the 5' end of the VH amplicon, and / or the VL amplicon and the VH amplicon are linked in step (iii) by a linker sequence encoding 15 to 25 amino acids; and / or 22. The method of embodiment 21, wherein the method further comprises the step of joining a sequence encoding an affinity tag to the VL / VH combination sequence obtained in step (iii).

[0290] 30. The method according to embodiment 21 to 22, further comprising the step of introducing an LCDR3 diversity module encoding a partially or completely random amino acid sequence for LCDR3 and / or an HCDR3 diversity module encoding a partially or completely random amino acid sequence for HCDR3 into the VL and / or VH sequences of the combination sequence, preferably into the sequences identified in step xv.

[0291] 31. The method according to embodiments 21 to 23, comprising the step of introducing a VL maturation module encoding partially or completely random amino acid sequences for LCDR1 and LCDR2, and / or a VH maturation module encoding partially or completely random amino acid sequences for HCDR1 and HCDR2, into the VL and / or VH sequences of the combined sequence, preferably into the sequences identified in step xv.

[0292] 32. The method of any one of embodiments 21 to 24, wherein step xi) comprises introducing the feline constant light part of the light chain (CL-lambda) and the constant heavy chain region (CH1) into an expression system such that in the expression system the VL polypeptide is expressed C-terminally linked CL-lambda and the VH polypeptide is expressed C-terminally linked CH1.

[0293] 33. A polypeptide library or a collection of nucleic acids obtainable or obtainable by the methods according to embodiments 21 to 25.

[0294] References Publication Catalog Baldwin, CI; Denham, DA (1994): Isolation and characterization of three subpopulations of IgG in the common cat (Felis catus). In Immunology 81 (1), pp. 155-160. Cho,K.; Youn,H.Y.; Okuda,M.; Satoh,H.; Cevario,S.; O’Brien,S.J.et al.(1998): Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes.In Immunogenetics 47 (3),pp.226-233.DOI: 10.1007 / s002510050352. Doki,Tomoyoshi; Takano,Tomomi; Hohdatsu,Tsutomu (2016): Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha.In The Journal of veterinary medical science 78 (9),pp.1447-1455.DOI: 10.1292 / jvms.16-0020. Ecker,Dawn M.; Jones,Susan Dana; Levine,Howard L.(2015): The therapeutic monoclonal antibody market.In mAbs 7 (1),pp.9-14.DOI: 10.4161 / 19420862.2015.989042. Enomoto,Masataka; Mantyh,Patrick W.; Murrell,Joanna; Innes,John F.; Lascelles,B.Duncan X.(2019): Anti-nerve growth factor monoclonal antibodies for the control of pain in dogs and cats.In The Veterinary record 184 (1),p.23.DOI: 10.1136 / vr.104590. Gearing,D.P.; Huebner,M.; Virtue,E.R.; Knight,K.; Hansen,P.; Lascelles,B.D.X.et al.(2016): In Vitro and In Vivo Characterization of a Fully Felinized Therapeutic Anti-Nerve Growth Factor Monoclonal Antibody for the Treatment of Pain in Cats.In Journal of veterinary internal medicine 30 (4),pp.1129-1137.DOI: 10.1111 / jvim.13985. Gearing,David P.; Virtue,Elena R.; Gearing,Robert P.; Drew,Alexander C.(2013): A fully caninised anti-NGF monoclonal antibody for pain relief in dogs.In BMC veterinary research 9,p.226.DOI: 10.1186 / 1746-6148-9-226. Gruen,M.E.; Thomson,A.E.; Griffith,E.H.; Paradise,H.; Gearing,D.P.; Lascelles,B.D.X.(2016): A Feline-Specific Anti-Nerve Growth Factor Antibody Improves Mobility in Cats with Degenerative Joint Disease-Associated Pain: A Pilot Proof of Concept Study.In Journal of veterinary internal medicine 30 (4),pp.1138-1148.DOI: 10.1111 / jvim.13972. Kanai,Tomoko Hosokawa; Ueda,Susumu; Nakamura,Toshihiro (2000): Identification of two allelic IgG1 Ch coding regions (Cγ1) of cat.In Veterinary immunology and immunopathology 73 (1),pp.53-62.DOI: 10.1016 / S0165-2427(99)00152-X. Kaplon,Helene; Reichert,Janice M.(2021): Antibodies to watch in 2021.In mAbs 13 (1),p.1860476.DOI: 10.1080 / 19420862.2020.1860476. Klotz,F.W.; Gathings,W.E.; Cooper,M.D.(1985): Development and distribution of B lineage cells in the domestic cat: analysis with monoclonal antibodies to cat mu-,gamma-,kappa-,and lambda-chains and heterologous anti-alpha antibodies.In Journal of immunology (Baltimore,Md.: 1950) 134 (1),pp.95-100. Olivieri,David N.; Haeften,Bernardo von; Sanchez-Espinel,Christian; Gambon-Deza,Francisco (2014): The immunologic V-gene repertoire in mammals.In bioRxiv,p.2667.DOI: 10.1101 / 002667. Raybould,Matthew I.J.; Marks,Claire; Krawczyk,Konrad; Taddese,Bruck; Nowak,Jaroslaw; Lewis,Alan P.et al.(2019): Five computational developability guidelines for therapeutic antibody profiling.In Proceedings of the National Academy of Sciences of the United States of America 116 (10),pp.4025-4030.DOI: 10.1073 / pnas.1810576116. Schultz,R.D.; Scott,F.W.; Duncan,J.R.; Gillespie,J.H.(1974): Feline immunoglobulins.In Infection and immunity 9 (2),pp.391-393.DOI: 10.1128 / iai.9.2.391-393.1974. Sitnikova,T.; Su,C.(1998): Coevolution of immunoglobulin heavy- and light-chain variable-region gene families.In Molecular biology and evolution 15 (6),pp.617-625.DOI: 10.1093 / oxfordjournals.molbev.a025965. Steiniger,Sebastian C.J.; Dunkle,William E.; Bammert,Gary F.; Wilson,Thomas L.; Krishnan,Abhiram; Dunham,Steven A.et al.(2014): Fundamental characteristics of the expressed immunoglobulin VH and VL repertoire in different canine breeds in comparison with those of humans and mice.In Molecular immunology 59 (1),pp.71-78.DOI: 10.1016 / j.molimm.2014.01.010. Steiniger,Sebastian C.J.; Glanville,Jacob; Harris,Douglas W.; Wilson,Thomas L.; Ippolito,Gregory C.; Dunham,Steven A.(2017): Comparative analysis of the feline immunoglobulin repertoire.In Biologicals : journal of the International Association of Biological Standardization 46,pp.81-87.DOI: 10.1016 / j.biologicals.2017.01.004. Tiller,Thomas; Waldhuber,Markus; Strohner,Ralf; Ladetzki,Baehs Kathrin; Prassler,Josef (6 / 21 / 2018): CANINE ANTIBODY LIBRARIES.Applied for by MORPHOSYS AG [DE] on 6 / 21 / 2018.App.no.US201816624514 20180621.Patent no.US2020223904 (A1).C07K16 / 00;C07K16 / 18.Priority no.EP20170177322 20170622;WO2018EP66563 20180621. Wabl,Matthias (5 / 23 / 2017): TRANSGENIC MAMMALS AND METHODS OF USE THEREOF.Applied for by TRIANNI INC [US] on 5 / 23 / 2017.App.no.US201715603334 20170523.Patent no.US2017306352 (A1).C12N15 / 85;A01K67 / 027;C07K16 / 46.Priority no.US201715603334 20170523;US201313818184 20130506;WO2011US45333 20110726;US20100367809P 20100726;US201662340234P 20160523. Walters,Rodney R.; Boucher,Joseph F.; Toni,Flavia de (2021): Pharmacokinetics and Immunogenicity of Frunevetmab in Osteoarthritic Cats Following Intravenous and Subcutaneous Administration.In Frontiers in veterinary science 8,p.687448.DOI: 10.3389 / fvets.2021.687448. * * *

Claims

1. and at least one VL / VH polypeptide member combination, wherein said at least one VL / VH polypeptide member combination is selected from the group consisting of SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:77 and SEQ ID NO:142, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:79 and SEQ ID NO:144, SEQ ID NO:80 and SEQ ID NO:145, SEQ ID NO:81 and SEQ ID NO:146, SEQ ID NO:82 and SEQ ID NO:147, SEQ ID NO:83 and SEQ ID NO:148, SEQ ID NO:84, and and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:87 and SEQ ID NO:152, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:89 and SEQ ID NO:154, SEQ ID NO:90 and SEQ ID NO:155, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:93 and SEQ ID NO:158, SEQ ID NO:94 and SEQ ID NO:159, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:96 and SEQ ID NO:161, SEQ ID NO:97 and SEQ ID NO:162, SEQ ID NO:98 and SEQ ID NO:163, SEQ ID NO:99 and SEQ ID NO:16 4, SEQ ID NO: 100 and SEQ ID NO: 165, SEQ ID NO: 101 and SEQ ID NO: 166, SEQ ID NO: 102 and SEQ ID NO: 167, SEQ ID NO: 103 and SEQ ID NO: 168, SEQ ID NO: 104 and SEQ ID NO: 169, SEQ ID NO: 105 and SEQ ID NO: 170, SEQ ID NO: 106 and SEQ ID NO: 171, SEQ ID NO: 107 and SEQ ID NO: 172, SEQ ID NO: 108 and SEQ ID NO: 173, SEQ ID NO: 109 and SEQ ID NO: 174, SEQ ID NO: 110 and SEQ ID NO: 175, SEQ ID NO: 111 and SEQ ID NO: 176, SEQ ID NO: 112 and SEQ ID NO: 177, SEQ ID NO: 113 and SEQ ID NO: 178, SEQ ID NO: 114 and SEQ ID NO:179, SEQ ID NO:115 and SEQ ID NO:180, SEQ ID NO:116 and SEQ ID NO:181, SEQ ID NO:117 and SEQ ID NO:182, SEQ ID NO:118 and SEQ ID NO:183, SEQ ID NO:119 and SEQ ID NO:184, SEQ ID NO:120 and SEQ ID NO:185, SEQ ID NO:121 and SEQ ID NO:186, SEQ ID NO:122 and SEQ ID NO:187, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:124 and SEQ ID NO:189, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:126 and SEQ ID NO:191, SEQ ID NO:127 and SEQ ID NO:192, SEQ ID NO:128 and SEQ ID NO:193,A synthetic feline antibody library comprising a combination selected from SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:130 and SEQ ID NO:195, SEQ ID NO:131 and SEQ ID NO:196, SEQ ID NO:132 and SEQ ID NO:197, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, SEQ ID NO:135 and SEQ ID NO:200, SEQ ID NO:136 and SEQ ID NO:201, and / or SEQ ID NO:137 and SEQ ID NO:202, or sequences having at least 95% sequence identity thereto.

2. SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:74 and SEQ ID NO:139, SEQ ID NO:75 and SEQ ID NO:140, SEQ ID NO:76 and SEQ ID NO:141, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:84 and SEQ ID NO:149, SEQ ID NO:85 and SEQ ID NO:150, SEQ ID NO:86 and SEQ ID NO:151, SEQ ID NO:88 and SEQ ID NO:153, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:95 and SEQ ID NO:160, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:112 and SEQ ID NO:177, 2. The synthetic feline antibody library of claim 1, comprising at least one VL / VH polypeptide member combination selected from sequence number 119 and SEQ ID NO:184, SEQ ID NO:123 and SEQ ID NO:188, SEQ ID NO:125 and SEQ ID NO:190, SEQ ID NO:128 and SEQ ID NO:193, SEQ ID NO:129 and SEQ ID NO:194, SEQ ID NO:133 and SEQ ID NO:198, SEQ ID NO:134 and SEQ ID NO:199, and / or SEQ ID NO:135 and SEQ ID NO:200, or a sequence having at least 95% sequence identity thereto.

3. A synthetic cat antibody library as described in claim 2, comprising at least one VL / VH polypeptide member combination selected from SEQ ID NO:73 and SEQ ID NO:138, SEQ ID NO:78 and SEQ ID NO:143, SEQ ID NO:91 and SEQ ID NO:156, SEQ ID NO:92 and SEQ ID NO:157, SEQ ID NO:110 and SEQ ID NO:175, SEQ ID NO:112 and SEQ ID NO:177, and / or SEQ ID NO:128 and SEQ ID NO:193, or a sequence having at least 95% sequence identity to said sequences.

4. A synthetic cat antibody library described in any one of claims 1 to 3, wherein at least one VL / VH polypeptide member combination lacks a post-translational modification (PTM) site.

5. A synthetic cat antibody library described in any one of claims 1 to 3, wherein the HCDR3 and / or LCDR3 regions of all VL / VH polypeptide member combinations are flanked by unique restriction enzyme sites.

6. A synthetic feline antibody library comprising at least one scFv or Fab antibody fragment, wherein said at least one scFv or Fab antibody fragment comprises any of the VL / VH polypeptide member combinations of any one of claims 1 to 3.

7. Substantially all VL / VH polypeptide member combinations are a) has a monomer content of at least 85% when expressed in Fab format in E. coli; and / or b) has a monomer content of at least 90% when expressed in an IgG format in a mammalian system; and / or c) A synthetic cat antibody library according to any one of claims 1 to 3, characterized by a Tm of 60°C or higher.

8. The synthetic feline antibody library of any one of claims 1 to 3, wherein the antibodies in the library are feline antibodies selected from single-chain Fv (scFv) fragments, Fab fragments, and F(ab)2 fragments.

9. A collection of nucleic acid molecules encoding the synthetic feline antibody library members of any one of claims 1 to 3; or a collection of vectors comprising said nucleic acid molecules; or A recombinant host cell comprising said nucleic acid molecule or said vector.

Images