WO2010130824A2

WO2010130824A2 - Collections and uses thereof

Info

Publication number: WO2010130824A2
Application number: PCT/EP2010/056637
Authority: WO
Inventors: Markus Enzelberger; Josef Prassler; Stefanie Urlinger
Original assignee: Morphosys Ag
Priority date: 2009-05-15
Filing date: 2010-05-14
Publication date: 2010-11-18
Also published as: WO2010130824A3

Abstract

The present disclosure relates to synthetic collections of candidate proteinaceous binding molecules, for example, antibodies, wherein one or more candidate proteinaceous binding molecules comprise an optimized property as compared to a parental proteinaceous binding molecule. In particular, the collections of candidate proteinaceous binding molecules are made by gene synthesis, and are based upon the sequence of the parental proteinaceous binding molecule, for example, antibody, therefore a custom collection of candidate proteinaceous binding molecules can be made specifically for one parental proteinaceous binding molecules. The present disclosure also relates to methods of making and using such collections for optimizing parental proteinaceous binding molecules, for example, antibodies. The present disclosure also relates to the candidate proteinaceous binding molecules of the synthetic collections comprising optimized properties.

Description

Collections and Uses Thereof

Cross-reference to related application

This application claims the benefit of U.S. provisional application serial number 61/291,346 filed December 30, 2009, U.S. provisional application serial number 61/184,338 filed June 5, 2009 and U.S. provisional application serial number 61/178,507 filed May 15, 2009, which are incorporated by reference in their entireties.

Field

The present disclosure relates generally to synthetic collections of candidate proteinaceous binding molecules, wherein one or more candidate proteinaceous binding molecules comprise an optimized property as compared to a parental proteinaceous binding molecule. The present disclosure relates more particularly to synthetic collections of candidate antibodies or functional fragments thereof, wherein one or more candidate antibodies comprise an optimized property as compared to a parental antibody. In particular, the collections of candidate proteinaceous binding molecules are made by gene synthesis, and are based on or derived from the sequence of a parental proteinaceous binding molecule, for example, antibody, therefore, a custom collection of candidates can be tailored to one parental proteinaceous binding molecule, for example, an antibody. The present disclosure also relates to methods of making and using such collections for optimizing parental proteinaceous binding molecules, such as, antibodies. The present disclosure also relates to the candidate proteinaceous binding molecules themselves, which may comprise an optimized property.

Background

Proteinaceous binding molecules, such as, peptides, or antibodies for diagnostic and therapeutic purposes are often identified via the use of libraries, such as phage, ribosomal or yeast display, which are screened against a specific immunogen. Often antibodies specific for an immunogen or antigen are identified, but have suboptimal properties. The problem that exists in the art is the identification of new, more effective methods of optimizing the properties of these parental antibodies. Methods have been described for introducing diversity into parental antibodies and selecting antibodies with optimized properties, such as, targeted mutagenesis, random mutagenesis by PCR based approaches, chain shuffling, complementarity determining region ("CDR") walking or cassette mutagenesis.

In maturation by targeted mutagenesis, the structure of a certain antibody or fragment thereof is resolved either by X-ray crystallography, or by protein design and molecular modeling, for example. Subsequently, residues which are assumed to be unfavorable are removed and/or new residues within sites that contact that immunogen are introduced, and the resulting mutants are characterized in detail. Such models have led to moderate affinity improvements, but also have generated mutants which completely lose immunogen binding (Wong et al., 1995; Riechmann et al., 1992). Others have introduced point mutations in defined positions within the CDRs without having any structural information from the parental antibody and characterized dozens of clones separately, some of which showed up to ten-fold improved affinity (Brummell et al., 1993). Wu et al. (1998) performed codon-based mutagenesis where every position of all six CDRs was exchanged for all twenty amino acids. This resulted in "only" 2592 variants each containing a single mutation, which were synthesized and screened. In a subsequent step, individual mutations were combined to further enhance antibody affinity reaching a final affinity improvement of up to 90-fold. This targeted mutagenesis approach is very time consuming and cumbersome. An additional drawback is the lack of diversity of matured candidates, as generally, only a few thousand matured candidates can be generated.

Another approach is random mutagenesis by error-prone PCR and the selection of affinity- improved antibodies by phage display (Hawkins et al., 1992; Gram et al., 1992). In addition, Boder et al. (2000) mutagenized a complete scFv sequence by error-prone DNA shuffling and selected antibodies with 10000-fold improved dissociation constants using flow cytometric cell sorting of yeast surface-displayed antibodies. The randomization of antibodies was successfully performed in vivo using either E. coli mutator cells (Coia et al., 2001) or even hypermutating B-cell lines (Cumbers et al., 2002). However, many of these methods are hampered by the fact that mutations are introduced at random, therefore, many of the antibodies generated are no longer specific for the immunogen.

Another way to generate modified and potentially improved successors of parental antibodies is "chain shuffling". Thereby, new combinations of heavy and light chains are made by recombining a single heavy or light chain with a library of partner chains (Marks et al, 1992; Schier et al, 1996a; Thompson et al, 1996; Hoogenboom et al, U.S. Patent No. 5,565,332). A drawback to this approach is that the new chain partners are often not compatible with the parental heavy or light chain; therefore, many of the antibodies generated are no longer specific for the immunogen.

Other approaches, such as, CDR walking and cassette mutagenesis limit the diversity introduced to only the CDR regions. For example, Yang et al (1995) generated libraries by saturation mutagenesis of the CDRs and selected affinity- improved antibodies by phage display. The sequential mutagenesis of up to five CDRs lead to an almost 400-fold affinity improvement. In cassette mutagenesis, pre-built CDR cassettes for all six CDR regions are provided, which have a variability of roughly 10⁴ up to 10⁹ members according to the length and the diversity of the respective CDRs. (Knappik et al (2000). These cassettes mimic the compositions of the CDRs of human rearranged antibody nucleic acids. By standard library cloning procedures the respective CDRs of the parental Fabs are exchanged for the corresponding maturation cassettes, thus introducing a high diversity within the chosen CDR region. A drawback of these approaches is that they lack customization based upon the unique parental antibody or fragment thereof. As demands for superior antibodies continue to increase, new maturation/optimization techniques are required.

The present application solves the problems of the above mentioned maturation approaches in the prior art and provides a novel multi- facetted solution which can be customized to any proteinaceous binding molecule whose properties need to be improved or changed.

Summary

The present disclosure provides a collection of candidate proteinaceous binding molecules, for example, an antibody, generated by gene synthesis, for example, by total gene synthesis, taking into account the particular nucleic acid or amino acid sequence of a parental proteinaceous binding molecule. This approach allows for a customized collection of candidate proteinaceous binding molecules to be generated for any parental proteinaceous binding molecule, wherein one or more candidate proteinaceous binding molecules has an optimized property as compared to the parental proteinaceous binding molecule. In particular aspects, said collection of candidate proteinaceous binding molecules is a maturation collection. In certain aspects, the proteinaceous binding molecule is an antibody or a functional fragment thereof. In certain aspects, the optimized property includes, but is not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In one aspect of the invention, a collection of candidate proteinaceous binding molecules is designed or generated in silico, wherein said candidate proteinaceous binding molecules are based on or derived from a parental proteinaceous binding molecule. The disclosed in silico design allows for almost unlimited customization or diversification of sequences, for example, within the variable domains, more specifically within the framework domains and/or the complementarity determing regions of the candidate proteinaceous binding molecules.

The collection of candidate proteinaceous binding molecules, once synthesized, represents the maturation collection, which is then screened against the immunogen of interest, wherein one or more candidate proteinaceous binding molecules is identified that comprises an optimized property as compared to the parental proteinaceous binding molecule.

The disclosed collections of candidate proteinaceous binding molecules comprise sequences, whether amino acid or DNA, which are based on or derived from a parental proteinaceous binding molecule. In some embodiments, the collection of candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. In some embodiments, the collection of candidate antibodies or functional fragments thereof comprise framework ("FR") and/or one or more complementarity determining regions ("CDR") that are substantially identical to, or based on or derived from the parental antibody or functional fragment thereof. Substantially identical to or based on or derived from allows for modifications within the framework and/or complementarity determining regions that result in an optimized property as compared to the parental proteinaceous binding molecule that includes, but is not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In some embodiments, the candidate antibodies or functional fragments thereof comprise framework and/or complementarity determining regions that are identical to the framework and/or complementarity determining regions of the parental antibody. In some embodiments, the candidate antibodies or functional fragments comprise framework and/or complementarity determining regions that are identical to the parental antibody or functional fragment thereof, wherein one or more complementarity determining regions are diversified, wherein the diversified complementarity determining regions result in one or more candidate antibodies or functional fragments thereof comprising an optimized property, wherein said property includes, but is not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In some embodiments, the candidate antibodies or functional fragments comprise framework and/or complementarity determining region sequences that are germline sequences which comprise the nearest germline family or gene to the framework and/or complementarity determining regions of the parental antibody.

In one aspect of the invention, the present disclosure provides a collection of candidate antibodies or functional fragments thereof comprising variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites. As standard cloning techniques are generally facilitated with the use of restriction sites, embodiments of the present disclosure provide synthetically generated nucleic acid sequences encoding candidate antibodies or functional fragments thereof wherein the nucleic acid sequences encoding the variable domains of the candidate antibodies or functional fragments thereof comprise a restriction site at the 5' and 3' ends. In certain aspects, it is advantageous to maintain the CDR3, specifically the HCDR3, of the parental antibody or functional fragment thereof, for example, to maintain binding specificity. In order to enable cloning of the CDR3 of the parental antibody or functional fragment thereof into the collection of candidate antibodies or functional fragments thereof, additional restriction sites may be necessary, therefore, embodiments of the present disclosure provide candidate antibodies or functional fragments thereof comprising variable regions, wherein the nucleic acids encoding the variable regions comprise a restriction site at the 5' end of the CDR3 region.

In other aspects, the present disclosure enables a kit comprising sequence data illustrating the sequences of a collection of candidate proteinaceous binding molecules or functional fragments thereof, or a design for sequences to be utilized to generate such a collection, wherein said sequence data are on a readable medium.

In other aspects, the present disclosure enables methods of producing, generating or making an isolated collection of candidate antibodies or functional fragments, comprising identifying a parental antibody or functional fragment thereof specific for an immunogen; identifying the nucleic acid sequence encoding the parental antibody or functional fragment thereof; generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments are substantially identical to, or based on or derived from the sequence of the parental antibody or functional fragment thereof; and synthesizing the collection of candidate antibodies or functional fragments thereof.

In other aspects, the present disclosure enables methods of identifying one or more candidate antibodies or functional fragments thereof having an optimized property, comprising the steps of identifying a parental antibody or functional fragment thereof specific for an immunogen; identifying the nucleic acid sequence encoding the parental antibody or functional fragment thereof; generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof are substantially identical to, or based on or derived from the sequence of a parental antibody or functional fragment thereof; synthesizing the collection of candidate antibodies or functional fragments thereof; screening the candidate antibodies or functional fragments thereof against said immunogen; identifying one or more candidate antibodies or functional fragments thereof comprising an optimized property, wherein said property is optimized compared to the property of the parental antibody or functional fragment thereof.

Detailed Description

The present disclosure relates to synthetic collections of candidate proteinaceous binding molecules or functional fragments thereof having an optimized property as compared to a parental proteinaceous binding molecule. In particular, the collections of candidate proteinaceous binding molecules are made by gene synthesis, and the sequences of the candidate proteinaceous binding molecules are based on or derived from the sequence of a parental proteinaceous binding molecule. Therefore, a custom collection of candidate proteinaceous binding molecules can be made specifically from or for one parental proteinaceous binding molecule. In some aspects, the proteinaceous binding molecules are antibodies or functional fragments thereof. In some aspects, the collections of candidate proteinaceous binding molecules comprise sequences substantially identical to, or based on or derived from the sequence of a parental proteinaceous binding molecule or functional fragment thereof, wherein the candidate proteinaceous binding molecules have an optimized property, which includes, but is not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

DEFINITIONS:

The term "collection" means an entity comprising at least two members. Such entity, includes, but is not limited to candidates comprising nucleic acids or amino acids or proteinaceous binding molecules or functional fragments thereof or antibodies or functional fragments thereof. A collection also may be in silico meaning that the collection comprises a list or database of sequences, whether DNA or amino acid. In the case the collection is in silico, it may also be a design for a collection. In this case, the design, would not include a list or database of all the sequences of the collection, but would include a design which would direct the synthesis of the collection. A collection also means a library.

The term "proteinaceous binding molecule(s)" refers to a molecule comprising at least two amino acids linked to each other by a peptide bond. The term "proteinaceous binding molecule" includes, but is not limited to antibodies, functional antibody fragments, enzymes, receptors, cytokines, hormones, transcription factors, signaling molecules, peptides, affϊbodies, or peptide aptamers.

The term "antibody" or "antibodies" includes, but is not limited to polyclonal, affinity- purified polyclonal, monoclonal, fully human, murine or rodent, chimeric, camelid or humanized antibodies or non-human antibodies. An antibody may be bivalent, trivalent, tetravalent or multivalent. An antibody may be monospecific, bispecific, trispecific or multispecific. An antibody may be monomeric, dimeric, trimeric, tetrameric or multimeric. An antibody may belong to any of the antibody classes, such as IgG, IgGl, IgG2, IgG3, IgG4, IgA (including human subclasses IgAl and IgA2), IgD, IgE, IgG, or IgM.

The term "functional fragment" of an antibody includes, but is not limited to any portion of an antibody which has a particular function, e.g. binding of immunogen, or antigen. Functional antibody fragments include antibody-like molecules which comprise antibody fragments combined with non-antibody structural scaffolds or linkers. Examples of functional antibody fragments are Fab, F(ab')2, Fab', Fv, scFv, single chains which include an Fc portion, chemically or genetically conjugated Fab fragments such as Fab₂ or Fab3, bis- scFv, diabody, minibody, triabody, tetrabody, unibody where a hinge region of an antibody is removed, and nanobodies. Examples of functional antibody fragments also include camelid derived VHH antibody fragments, cartilaginous- fish derived VNAR antibody fragments or other antibody fragments derived from non-human origin. Examples of functional antibody fragments include antibody formats such as SMIP (SMIP contains a binding domain, a hinge domain and an effector domain) and Scorpion (multimers of SMIP), or DVD Ig. Additionally, functional antibody fragments are often engineered to include new functions or properties.

The term "candidate proteinaceous binding molecule(s)" means either a nucleic acid sequence encoding a proteinaceous binding molecule, or a proteinaceous binding molecule comprising an amino acid sequence, wherein the sequence of the candidate is modified as compared to the sequence of the parental proteinaceous binding molecule, which modifications include DNA and/or amino acid modifications which may result in an optimized property as compared to said parental proteinaceous binding molecule, including, but not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

The term "candidate antibody" or "candidate antibodies" means either a nucleic acid sequence encoding an antibody, or antibodies in plural, or an antibody or antibodies in plural, comprising an amino acid sequence, wherein the sequence of the candidate(s) is modified as compared to the sequence of the parental antibody, which modifications include DNA and/or amino acid modifications which may result in an optimized property as compared to said parental antibody, including, but not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

The term "based on or derived from" means that the sequence, either DNA and/or amino acid, of the parental proteinaceous binding molecule or antibody or functional fragment thereof is known, or identified and the sequence, either DNA and/or amino acid, of the candidates, whether proteinaceous binding molecules or antibodies or functional fragments thereof, are modifications of the parental proteinaceous binding molecule or antibodies or functional fragments thereof, which modifications include DNA and/or amino acid modifications which may result in an optimized property as compared to said parental proteinaceous binding molecule or antibody or functional fragment thereof including, but not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets. For example, the candidate proteinaceous binding molecules or antibodies or functional fragments thereof may comprise portions with the same sequence as the parental proteinaceous binding molecule or antibody or functional fragment thereof, for example, framework and/or complementarity determining regions or comprise framework and/or complementarity determining regions of the nearest germline family or gene to the parental proteinaceous binding molecule or antibody or functional fragment thereof.

The term "synthesis" or "synthesized" means gene synthesis, where nucleic acid sequences are synthesized into physical DNA, comprising polynucleotides. Generally, the sequences of the nucleic acids of the present disclosure are determined in silico, and the sequence data is synthesized into physical DNA. Standard DNA synthesis comprises single nucleotide synthesis, where single-stranded oligo-nucleotides are generated and then the overlapping oligonucleotides are ligated using a PCR-like assembly. Companies, such as, Sloning (Puchheim, Germany), Geneart (Regensburg, Germany), DNA2.0 (Menlo Park, CA USA), and Genscript (Piscataway, NJ USA) provide gene synthesis technology. Sloning, for example, utilizes a library of pre-made double stranded triplet nucleotides, which are subsequently ligated.

The term "synthetic" describes a molecule that is made by synthesis or synthesized. For example, a synthetic nucleic acid may be generated by gene synthesis. In addition, when the nucleic acid is synthetic then the proteinaceous binding molecule or a functional fragment thereof, for example, antibody, that is encoded by the nucleic acid is also synthetic.

The term "sequence" means the sequence of two or more nucleic acids or amino acids. Sequence or "sequence data" includes nucleic acids or amino acids, in silico, or the letters themselves that designate nucleic acids or amino acids. "Substantially devoid of restriction sites" means that a nucleic acid molecule encoding a proteinaceous binding molecule, such as, an antibody or a functional fragment thereof may not contain more restriction sites than required for a given purpose. As the case may be, additional restriction sites may, however, be advantageous for additional methods or uses. It is known that restriction sites exist naturally within any given nucleic acid sequence, and that these naturally occurring restriction sites cannot be removed. The restriction sites described herein refer to restrictions sites, which are either introduced into the sequence or if naturally occurring are subsequently used for cloning or exchange of regions of the variable region, for example, framework and/or complementarity determining regions. Where the proteinaceous binding molecule is an antibody or a functional fragment thereof, restriction sites are not present at the boundary between each framework region and complementarity determining region, but restriction sites may be present at the boundary between one or more framework regions and complementarity determining regions. Substantially devoid of restriction sites may also mean having one restriction site located in the area 5' of CDR3. Where a restriction site is located in the area 5' of CDR3 it may be utilized to clone the CDR3 region of a parental antibody into the maturation collection. In some embodiments, substantially devoid of restriction sites means that nucleic acids encoding antibodies or functional fragments thereof contain restriction sites at, or around, the 5' end and 3' end of the variable domains.

"Substantially identical to" describes a candidate proteinaceous binding molecule or antibody or functional fragment thereof having a nucleic acid or amino acid sequence with modifications as compared to a parental proteinaceous binding molecule or antibodies or functional fragments thereof. Substantially identical to includes modifications within the nucleic acid sequence encoding the frameworks and/or CDRs or amino acid sequence of the frameworks and/or CDRs, which result in optimized properties, which include, but are not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets. The term "structural/functional motifs or domains" means a region or a portion of a proteinaceous binding molecule or a functional fragment thereof that is directly or indirectly required for the desired biological property to be optimized. In one aspect, the term "structural/functional motifs or domains" means a region or a portion of a proteinaceous binding molecule or a functional fragment thereof that is specific for binding to a target molecule, for example, a complementarity determining region. In one aspect, the term "structural/functional motifs or domains" means a region or a portion of a proteinaceous binding molecule or a functional fragment thereof that is required for providing support to structural/functional motifs or domains specific for a target molecule. In one aspect, the term "structural/functional motifs or domains" means a region or a portion of a proteinaceous binding molecule or a functional fragment thereof that is required for humanization of proteinaceous binding molecules or functional fragments thereof. In one aspect, a proteinaceous binding molecule or a functional fragment thereof may contain one or more structural/functional motifs or domains.

The term "target molecule" means any molecule to which a proteinaceous binding molecule or a functional fragment thereof is capable of binding. The term "target molecule" includes, but is not limited to immunogens, antigens, receptors, toxins, drugs, substrates for enzymes, or signaling molecules. A target molecule can be a monosaccharide, a polysaccharide, a protein, a peptide, or lipid.

The term "variable chain/region/domain" includes, but is not limited to the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V_L (including Vk and Vλ), V_H, J_L (including Jk and Jλ), and J_H nucleic acids that make up the variable light chain (including K and λ) and variable heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (V_L and V_H) consists of a "framework" or "FR" region interspersed by three hypervariable regions referred to as "complementarity determining regions" or "CDRs." The extent of the framework region and CDRs have been precisely defined (see Kabat, 1991, J. Immunol, 147, 915-920.; Chothia & Lesk, 1987, J. MoI. Biol. 196: 901-917; Chothia et al, 1989, Nature 342: 877-883; Al- Lazikani et al., 1997, J. MoI. Biol. 273: 927-948). The framework regions of an antibody, that is the combined framework regions of the constitutent light and heavy chains, serves to position and align the CDRs, which are primarily responsible for binding to an antigen.

The term "member" means one individual species of a collection. The term "CDR variant" means a candidate comprising one or more amino acid residues within the CDR region that is different than the amino acid residues within the CDRs of the other candidates of a collection. A variant of the present disclosure is identified by comparing the nucleic acid sequences or amino acid sequences of each of the candidates of a collection. As the sequences are determined in silico, and the sequence data is located in a database, one can compare the sequences of each of the nucleic acids irrespective of the size of the collections.

The term "CDR diversification" or "diversified complementarity determing region" means the modification of the amino acid composition of a CDR by any suitable method, including those methods described herein. CDRs are generally known to be the immunogen binding regions, therefore, modifications within the CDRs may lead to optimized properties, including, but not limited to higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets. A collection may be more likely to include a candidate comprising an optimized property if one or more CDRs have a large diversity.

The term "parental proteinaceous binding molecule" means a single proteinaceous binding molecule that has been identified as specific for a target molecule or immunogen by methods known to one of skill in the art, wherein a proteinaceous binding molecule includes an antibody.

The term "parental antibody" means a single antibody, or a functional fragment thereof, that has been identified as specific for a target molecule or immunogen by methods known to one of skill in the art. A parental may be identified by screening a collection against a target molecule. The terms includes an antibody or functional fragment thereof that has sub-optimal properties, for which it is desirable to improve such properties, such as, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

The term "homologous" refers to portions or sections of a proteinaceous binding molecule which have conserved or similar residues at corresponding positions in their primary, secondary or tertiary structure. This includes antibodies or functional fragments thereof having the conserved or similar residues at a corresponding position in their primary, secondary or tertiary structure. The term also extends to two or more nucleotide sequences encoding homologous polypeptides. Homologous also includes inter-species homology where portions of an antibody or fragment thereof, for example, a murine antibody, can be replaced with homologous human amino acids sequences.

The term "immunogen" means any target molecule to which proteinaceous binding molecule, for example, an antibody, or a functional fragment thereof, binds or to or is specific for. An immunogen may be an antigen, a pathogen or other infectious agent that can cause disease or illness in a host. An immunogen may be a protein or a peptide or may even be a nonpeptide entity, such as a sugar moiety or lipid. Said antibody, or functional fragment thereof, is preferably a therapeutic antibody. For the purposes of generating therapeutic antibodies, an immunogen is preferably a cell surface antigen, but may be any other type of antigen, including a soluble protein. An immunogen comprises at least one epitope orimmunogenic determinant. An immunogen may also contain several epitopes or immunogenic determinants. The epitope comprises the specific amino acid residues that are bound by a single protein binding moiety. An epitope can be linear, conformational or discontinuous. An epitope can also be defined as the specific amino acids that protein binding moieties can be generated against.

The term "optimized" or "matured" is used to describe a property that is enhanced or preferred as compared to the respective property of a parental proteinaceous binding molecule or a functional fragment thereof, for example, an antibody.

The term "optimized property" means any combination of the following: higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

The term "property" includes, but is not limited to, binding affinity for a specific immunogen; binding specificity for a specific immunogen; human characteristics; immunogenicity; stability, including thermal and/or serum stability; display rates in phage, bacterial, eukaryotic, including mammalian, display; expression levels in bacterial or eukaryotic, including mammalian, cells; tendency for aggregation; presence of potential T- cell epitopes; deviations from germline; presence of undesired amino acid residues, such as methionines or cysteines; presence of potential glycosylation sites; cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets. If the term "property" is used in the context of other proteinaceous binding molecules, then such property might include any property or effect of a respective proteinaceous binding molecule. Non-limiting examples include the catalytic activities of enzymes, such as the conversion rate or the activity, the heat or cold stability of enzymes or protein, resistances to detergents or other organic or inorganic matter, or any other related property.

"Human characteristics" means having a portion of nucleic acids or amino acid sequences recognized as human. Optimized human characteristics includes increasing the number of human amino acids in a proteinaceous binding molecule or a functional fragment thereof, for example, an antibody, or increasing the proportion of human versus non- human amino acids in a proteinaceous binding molecule or a functional fragment thereof, for example, an antibody. In one aspect, optimized human characteristics includes increasing the number of human amino acids in an antibody or a fragment thereof, or increasing the proportion of human versus non- human amino acids in an antibody or a fragment thereof.

The germline families and genes of the V_H (Tomlinson, 1992), Vλ (Ignatovich, 1997) and VK (Schable, 1993) have been disclosed in the respective publications. The term "germline framework regions" or "germline complimentarity determining regions" describes the specific regions of the V_H and/or V_L that are germline in sequence. The sequence of the respective germline families and genes are described in the following websites: http://www.imgt.org/textes/IMGTrepertoire/Proteins/taballeles/human/IGH/IGHV/Hu_IGHV all.html, or at vbase: http://vbase.mrc-cpe.cam.ac.uk/ or at Dr. Annemarie Honegger's website at http://www.bioc.uzh.ch/antibody/Sequences/index.html.

"Non-human sequence(s)" includes nucleic acid or amino acid sequences that are of non-human origin, for example, nucleic acid or amino acid sequences from rodent, for example, murine or rat, rabbit, or camel or cartilaginous fish.

"Restriction sites" are specific sequences of nucleotides that are recognized by restriction enzymes (restriction endonucleases), which cut the nucleic acid molecules at sequences specific for a given restriction enzyme. Restriction sites may occur naturally or may be added, deleted, modified or otherwise manipulated by genetic methods known to the skilled artisan. In certain aspects of the present invention restriction sites may be added or removed which render the respective nucleaic acids molecules more germline- like.

"Readable medium" means an electronic document, such as one stored on a computer, or on an electronic storage device, for example, a CD or flash drive, or a hard document, such as paper.

In one aspect, the collections of candidate proteinaceous binding molecules are based on or derived from the sequence of a parental proteinaceous binding molecule and are generated entirely or substantially in silico and then synthesized into physical DNA.

In some aspects, the present disclosure provides methods of making, generating or producing synthetic collections of candidate antibodies or functional fragments thereof comprising the steps of synthesizing a collection of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof have at least one optimized property as compared to a parental antibody or functional fragment thereof. In some embodiments, the present disclosure provides methods of making, generating or producing a collection of candidate antibodies or functional fragments thereof having an optimized property compared to a parental antibody or functional fragment thereof, comprising the steps of: identifying a parental antibody or functional fragment thereof specific for an immunogen; identifying the nucleic acid sequence encoding the parental antibody or functional fragment thereof; generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. In some embodiments, the methods of making, generating or producing the present collections comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof are substantially identical to, or based on or derived from the sequences of the parental antibody or functional fragment thereof; and synthesizing the collection of candidate antibodies or functional fragments thereof.

In some embodiments, the methods of making, generating or producing the present collections comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions that are substantially identical to or based on or derived from the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions identical to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. In some embodiments, the one or more diversified complementarity determining regions result in candidate antibodies or functional fragments thereof comprising an optimized property as compared to said parental antibody or functional fragment thereof.

In some embodiments, the methods of making, generating or producing the present collections comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions substantially identical to or based on or derived from the framework and/or complementarity determining regions of the parental antibody or functional fragment thereof, wherein substantially identical to or based on or derived from describes modifications within the framework regions and/or complementarity determining regions that result in an optimized property antibody or functional fragment thereof.

In some embodiments, the methods of making, generating or producing the present collections comprise generating a collection, preferably in silico, of antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline family or gene to the framework regions and/or complementarity determining regions of the parental antibody. In some embodiments, the sequences of said candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. In some embodiments, the diversified complementarity determining regions result in candidates comprising an optimized property. In some embodiments, the parental antibody or functional fragment thereof comprises non- human sequences.

In some embodiments, the methods of making, generating or producing the present collections comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise V_H regions comprising one or more diversified complementarity determining regions and V_L regions substantially identical to or based on or derived from said parental antibody or a functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise V_L regions comprising diversified complementarity determining regions and V_H regions substantially identical to, or based on or derived from said parental antibody or functional fragment thereof. In some embodiments, candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising diversified complementarity determining regions.

In some aspects, the property to be optimized is selected from the group consisting of higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In other aspects, the present disclosure enables methods of identifying candidate proteinaceous binding molecules, for example, antibodies or functional fragments thereof, having an optimized property as compared to a parental proteinaceous binding molecule. In some embodiments, such methods comprise the steps of identifying a parental proteinaceous binding molecule specific for an immunogen; identifying the nucleic acid sequence encoding the parental proteinaceous binding molecule; generating a collection, preferably in silico, of candidate proteinaceous binding molecules, wherein the candidate proteinaceous binding molecules are substantially identical to, or based on or derived from the parental proteinaceous binding molecule; synthesizing the collection of candidate proteinaceous binding molecules; screening the candidate proteinaceous binding molecules against said immunogen; and identifying one or more candidate proteinaceous binding molecules comprising an optimized property, wherein said property is optimized compared to the property of the parental proteinaceous binding molecule.

In other aspects, the present disclosure enables methods of identifying a candidate antibody or functional fragment thereof having an optimized property compared to a parental antibody or functional fragment thereof, comprising the steps of: identifying a parental antibody or functional fragment thereof specific for an immunogen; identifying the nucleic acid sequence encoding the parental antibody or functional fragment thereof; generating a collection of candidate antibodies or functional fragments thereof, wherein said candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions; synthesizing the collection of candidate antibodies or functional fragments thereof; screening the candidate antibodies or functional fragments thereof against said immunogen; and identifying one or more candidate antibodies or functional fragments thereof comprising an optimized property, wherein said property is optimized compared to the property of the parental antibody or functional fragment thereof.

In some aspects, the methods of identifying candidate antibodies or functional fragments thereof comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise framework regions and/or one or more complementarity determining regions that are substantially identical to, or based on or derived from the framework regions and/or complementartiy determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise framework regions and/or complementartiy determining regions identical to the framework regions and/or complementartiy determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. In some embodiments, the one or more diversified complementarity determining regions result in candidates comprising an optimized property as compared to the parental antibody or functional fragment thereof.

In some embodiments, the methods of identifying candidate antibodies or functional fragments thereof comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise framework regions and/or one or more complementarity determining regions substantially identical to or based on or derived from the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof, wherein substantially identical to or based on or derived from allows for modifications within the framework and/or complementarity determining regions that result in an optimized property as compared to the parental antibody or functional fragment thereof.

In some embodiments, the methods of identifying candidate antibodies or functional fragments thereof comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline family or gene to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In these embodiments, a parental antibody or functional fragment thereof specific for an immunogen is identified, and the sequence of the parental antibody or functional fragment thereof is identified. As a next or additional step, the nearest germline family or gene of both the VH and VL of the parental antibody or functional fragment thereof is identified. In these embodiments, the methods of identifying candidate antibodies or functional fragments thereof further comprise identifying the nearest germline family or gene of the parental antibody or functional fragment thereof s variable regions, and comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof comprising the nearest germline family or gene of the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. Specifically, a CDR3, preferably HCDR3, of the parental may be maintained and inserted by standard cloning methods into the collection of candidates, and the collection of candidates may be generated, wherein the candidate antibodies or functional fragments thereof comprise one or more diversified CDRs, wherein the diversified CDRs could include CDRl, CDR2 or the CDR3 region not being maintained from the parent. In addition, the collection of candidate antibodies or functional fragments thereof may comprise germline framework regions or framework regions substantially similar to or based on or derived from the framework regions of the parental antibody or functional fragment thereof. In some embodiments, the diversified complementarity determining regions result in candidate antibodies or functional fragments thereof comprising an optimized property as compared to the parental antibody or functional fragment thereof. In some embodiments, the parental antibody or functional fragment thereof comprises non-human sequences.

In some embodiments, the property to be optimized is selected from the group consisting of higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In some embodiments, the methods of identifying candidate antibodies or functional fragments thereof comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise V_H regions comprising one or more diversified complementarity determining regions and V_L regions substantially identical to or based on or derived from said parental antibody or a functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified complementarity determining regions and V_H regions substantially identical to or based on or derived from said parental antibody or a functional fragment thereof. In some embodiments, said candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising one or more diversified complementarity determining regions.

In some embodiments, the methods of identifying candidate antibodies or functional fragments thereof comprise generating a collection, preferably in silico, of candidate antibodies or functional fragments thereof, wherein the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise restriction sites at the 5' and 3' ends. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise a restriction site at the 5' prime end of the CDR3 region. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise one or more restriction sites at the boundary of one or more framework regions and one or more complementarity determining regions, for example at the boundary of FRl and CDRl, CDRl and FR2, FR2 and CDR2, FR3 and CDR3, CDR3 and FR4. The additional restriction sites would facilitate cloning of a parental antibody or a functional fragment thereof 's CDR and/or FR region into the collections described herein and/or facilitate the cloning of a collection of CDR and/or FR region into the collections described herein. Portions of the parental antibody or a functional fragment thereof, such as CDRs of FRs may be desirable to maintain in order to maintain certain properties of the parental antibody or a functional fragment thereof, for example, binding specificity.

In one aspect, the present disclosure relates to an isolated collection of candidate antibodies or functional fragments thereof, preferably, antibodies or functional fragments thereof, wherein one or more candidate antibodies or functional fragments thereof comprises an optimized property as compared to the property of a parental antibody or a functional fragment thereof. In particular, the collections of candidate antibodies or functional fragments thereof are made by gene synthesis, wherein the candidate antibodies or functional fragments thereof are substantially identical to, or based on or derived from the sequences of the parental antibody or a functional fragment thereof, therefore a custom collection of candidate antibodies or functional fragments thereof can be made specifically for one parental antibody or a functional fragment thereof. In some aspects, the collections of candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions of a parental antibody or a functional fragment thereof, wherein the one or more candidate antibodies or functional fragments thereof have an optimized property as compared to the parental antibody or functional fragment thereof. The collections of candidate antibodies or functional fragments thereof comprise the FR sequences and one or more CDR sequences from a parental antibody or a functional fragment thereof, where one or more of the CDRs is diversified. More preferably, the collection of candidate antibodies or functional fragments thereof further comprise targeted modifications within the CDRs and/or FR regions in order to generate candidates having an optimized property as compared to a parental antibody or a functional fragment thereof.

In some embodiments, the isolated collection of candidate antibodies or functional fragments thereof comprises variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites, wherein said candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions.

In some embodiments, the isolated collection of candidate antibodies or functional fragments thereof comprises variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites, wherein said candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions substantially identical to or based on or derived from the framework regions and/or complementarity determining regions of a parental antibody or a functional fragment thereof, and wherein one or more of said candidate antibodies or functional fragments thereof comprises an optimized property, wherein said property is optimized compared to the property of a parental antibody or functional fragment thereof.

In some embodiments, the collections of candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions that are identical to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise one or more diversified CDR regions. In some embodiments, the diversified CDR regions result in candidate antibodies or functional fragments thereof comprising an optimized property as compared to the parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise framework regions and/or complementarity determining regions substantially identical to or based on or derived from the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof, wherein substantially identical to and based on or derived from allows for modifications within the framework regions and/or complementarity determining regions that result in an optimized property as compared to the parental antibody or functional fragment thereof.

In some embodiments, the collection of candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline family or gene to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof. In these embodiments, a parental antibody or functional fragment thereof specific for an immunogen is identified, and the sequence of the parental antibody or functional fragment thereof is identified. As a next or additional step, the nearest germline family or gene of both the VH and VL of the parental antibody or functional fragment thereof is identified. In some embodiments, the candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions. Specifically, either CDR3 of the parental antibody or functional fragment thereof may be maintained and inserted by standard cloning methods into the collection of candidate antibodies or functional fragments thereof, and the collection of candidate antibodies or functional fragments thereof may be generated, wherein the candidate antibodies or functional fragments thereof comprise one or more diversified CDRs, wherein the diversified CDRs may include CDRl, CDR2 or the CDR3 region not being maintained from the parent. In other embodiments, the collection of candidate antibodies or functional fragments thereof comprises germline framework regions and/or framework regions substantially similar to or based on or derived from the framework regions of the parental antibody or functional fragment thereof. In some embodiments, the diversified complementarity determining regions result in candidate antibodies or functional fragments thereof comprising an optimized property as compared to the parental antibody or functional fragment thereof.

In some embodiments, the collection of candidate antibodies or functional fragments thereof comprise one or more candidate antibodies or functional fragments thereof comprising an optimized property as compared to the property of a parental, wherein the property to be optimized is selected from the group consisting of higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In some embodiments, the parental antibody or functional fragment thereof comprises non-human sequences. In some embodiments, the collection of candidate antibodies or functional fragments thereof comprises V_H regions comprising one or more diversified CDR regions and V_L regions substantially identical to or based on or derived from said parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified CDR regions and V_H regions substantially identical to or based on or derived from said parental antibody or functional fragment thereof. In some embodiments, the candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising one or more diversified CDR regions. In some embodiments, the candidate antibodies or functional fragments thereof are encoded by nucleic acids comprising restriction sites on the 5' and 3' ends of the variable domains. In some embodiments, the candidate antibodies or functional fragments thereof are encoded by nucleic acids comprising a restriction site 5' of the CDR3 region.

In some embodiments, the collection of candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise restriction sites at the 5' and 3' ends. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise a restriction site at the 5 ' prime end of the CDR3 region. In some embodiments, the candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise one or more restriction sites at the boundary of one or more framework regions and one or more complementarity determining regions, for example at the boundary of FRl and CDRl, CDRl and FR2, FR2 and CDR2, FR3 and CDR3, CDR3 and FR4. The additional restriction sites would facilitate cloning of a parental antibody or functional fragment thereof CDR and/or FR region into the collections described herein and/or facilitate the cloning of a library of CDR and/or FR region into the collections described herein. Portions of the parental antibody or functional fragment thereof, such as CDRs of FRs may be desirable to maintain in order to maintain certain properties of the parental antibody or functional fragment thereof, for example, binding specificity.

The collections and methods of making and using the collections of the present invention for the first time allow for maturation collections, of for example, antibodies, that are tailored to features of one or more parental antibodies or functional fragments thereof from which they are based, which has several benefits, including substantially increasing the chance of finding candidate antibodies or functional fragments thereof, for example, antibodies, having properties desirable in drug development and human administration.

In a particular aspect of the invention, the present disclosure is the first to provide a maturation collection generated by gene synthesis, for example, total gene synthesis, taking into account the particular nucleic acid or amino acid sequence of the parental antibody or a functional fragment thereof to be matured. This approach allows for a customized collection of candidate antibodies or functional fragments thereof for a particular parental antibody or functional fragment thereof, wherein one or more candidate antibodies or functional fragments thereof has an optimized property as compared to the parental antibody or functional fragment thereof. In some embodiments, the disclosed collections of candidate antibodies or functional fragments thereof may comprise robust CDR diversity while at the same time allowing for FR and/or CDR customization, which may lead to an optimized property as compared to the parental antibody or functional fragment thereof. In some embodiments, one or more of the FRs and/or CDRs of the particular parental antibody or functional fragment thereof may be included in the collection of candidate antibodies or functional fragments thereof.

In one aspect of the invention, the present disclosure provides a maturation collection of at least 1 XlO candidate antibodies or functional fragments thereof comprising synthetically generated nucleic acid sequences encoding proteinaceous binding molecules wherein the candidate antibodies or functional fragments thereof sequences present in the collection are substantially devoid of restriction sites.

In some aspects, the present invention enables a substantially isolated collection of at least 1 x 10³ candidate antibodies or functional fragments thereof comprising synthesized nucleic acids encoding antibodies or functional fragments thereof, wherein said nucleic acids encoding the boundaries between each framework region and each complementary determining region are substantially devoid of restriction sites.

In other aspects, the present disclosure enables methods of producing, generating or making an isolated collection of at least 1 XlO³ candidates, comprising synthesizing a diverse collection of candidate proteinaceous binding molecules or functional fragments thereof.

In some embodiments, the present disclosure enables methods of producing, generating or making an isolated collection of at least 1 XlO³ candidates, comprising synthesizing a diverse collection of candidates, wherein said candidates comprising one or more diversified structural/functional motifs or domains. In some embodiments, the maturation collections of the present invention and the methods of making or using the same comprise candidates wherein the nucleic acids encoding the variable regions of the candidates are substantially devoid of restriction sites. In some embodiments the nucleic acids encoding the candidates comprise two restriction sites, namely, one at the 5' end and the other at the 3' end of the variable domains. For example, in the case of candidate antibodies or functional fragments thereof, a restriction site may be present at the 5' end and 3' end of the V_H and V_L domains. In some embodiments, the present disclosure provides collections of candidates and method of making and using the same, wherein the nucleic acids encoding the variable regions of said candidates comprise a restriction site 5' prime of the CDR3 region.

In some embodiments, the present disclosure enables collections of candidates, wherein each candidate comprises nucleic acids encoding a structural/functional motif or domain sequence of a proteinaceous binding molecule, wherein at least one structural/functional motif or domain sequence is unique within candidates in said collection.

Collections of the present disclosure, including the embodiments of the preceeding paragraphs, comprise at least IXlO³ candidates, preferably at least IXlO⁴ candidates, preferably at least 1OX⁵ candidates, preferably at least IXlO⁶ candidates, more preferably at least IXlO⁷ candidates, most preferably at least IXlO⁸ candidates, preferably at least IXlO⁹ candidates, preferably at least IXlO¹⁰ candidates, preferably at least IXlO¹¹ candidates, preferably IXlO¹² candidates, and preferably IXlO¹³ candidates.

The collections of candidates of the present disclosure are synthesized by gene synthesis. In some embodiments the V_H and/or V_L are synthesized without restriction sites between the framework regions and complementarity determining regions. In some embodiments the V_H and/or V_L are synthesized with one restriction site between a framework region and a complementarity determining region, for example between FR3 and CDR3 on either the V_H and/or V_L. Such a restriction site would allow for easy cloning of a single CDR3, for example, the CDR3 of the parental, or a CDR3 library into the collection. Additional restriction sites may be synthesized in the V_H and/or V_L between the framework regions and complementarity determining regions, for example, between FRl and CDRl, between CDRl and FR2, between FR2 and CDR2, between CDR2 and FR3 and/or between CDR3 and FR4.

In one aspect of the invention, a parental proteinaceous binding molecule is identified by any method known to one of skill in the art. Such methods include, but are not limited to phage display, yeast display, ribosomal display, transgenic mouse immunization, hybridoma technology or mouse immunization.

Once a parental proteinaceous binding molecule is obtained or identified, the nucleic acid sequence encoding the parental proteinaceous binding molecule may be determined. If the proteinaceous binding molecule is identified using phage display or other display technique then the DNA encoding the parental is readily available for sequencing. Otherwise, the DNA encoding the parental proteinaceous binding molecule can be isolated and sequenced.

In some embodiments, the present disclosure enables collections of candidate antibodies or functional fragments thereof, wherein each candidate antibodies or functional fragments thereof comprises a CDR sequence, wherein at least one CDR of each candidate antibodies or functional fragments thereof is unique within the candidate antibodies or functional fragments thereof in said collection. In such embodiments, the collections of candidate antibodies or functional fragments thereof based upon the parental antibody or functional fragment thereof comprises at least one diversified CDR, wherein each candidate antibodie or functional fragment thereof comprises at least one CDR that is unique within the candidate antibodies or functional fragments thereof in said collection.

In other embodiments of the present disclosure, synthetic collection of candidate antibodies or functional fragments thereof comprises framework regions that are the same or substantially the same amino acid sequence as the framework regions of a parental antibody or functional fragment thereof and comprises one or more CDR regions that are the same or substantially the same amino acid sequence as the CDR regions of the parental antibody or functional fragment thereof, wherein one or more of the other CDR regions are diversified, and wherein the collection has been synthesized according to the techniques herein. The CDR diversification of the present disclosure enhances the diversity of the collections of candidate antibodies or functional fragments thereof and increases the possibility of identifying candidate antibodies or functional fragments thereof having optimized properties. Methods of CDR diversification are known to one of skill in the art and may include Knappik et al. 2000; WO 97/08320, complete randomization of the CDRs, or custom CDR diversification by rational design.

Some embodiments of the present disclosure comprise collections of candidate antibodies or functional fragments thereof comprising either the heavy and/or light chain FR' s 1-4 and HCDR3 of the parental antibody or functional fragment thereof, wherein one or more of the following CDRs are diverisified, HCDRl, HCDR2, LCDR 1, LCDR 2 and/or LCDR 3. As the disclosed collections are generated by gene synthesis, and are thus fully customizable, one or more of the CDRs and/or FRs of the parental antibody or functional fragments thereof can be maintained and one or more of the CDRs and/or FRs of the parental antibody or functional fragment thereof can be diversified, therefore collection of the present disclosure may comprise, one or more of the CDRs and/or FRs of the parental antibody or functional fragment thereof, where one or more of the CDRs and/or FRs are diversified.

Some embodiments of the present disclosure comprise collections of candidate antibodies or functional fragments thereof comprising either the heavy and/or light chain FR' s 1-4 and HCDR3 and LCDRl of the parental antibody or functional fragment thereof, wherein, HCDRl and/or 2 and LCDR3 and/or 2 are diversified. In this embodiment, HCDR3 can be maintained so that each of the candidate antibodies or functional fragments thereof in the collection comprise the same specificity to an immunogen as the parental antibody or functional fragment thereof. In the V_H, either the HCDRl and/or 2 are diversified so as to generate candidate antibodies or functional fragments thereof of the collection having optimized binding affinity. In the same example, LCDRl and/or LCDR2 may be maintained, as they may have only a minor influence on the overall binding to an immunogen, therefore, diversification may not lead to optimized properties. The LCDR3 and/or LCDR2 are diversified in order to generate candidate antibodies or functional fragments thereof of the collection having an optimized property as compared to the parental antibody or functional fragment thereof. The above example, is not intended to be limiting, as the present disclosure enables one of skill in the art to provide collections comprising diversification in any of the CDR regions. This customization is made possible by generating the collections via total gene synthesis.

In some aspects, more than one collection may be synthesized in order to identify proteinaceous binding molecules, for example, antibodies, with optimized properties compared to a parental proteinaceous binding molecule. For example, more than one collection may be synthesized in order to identify candidate antibodies or functional fragments thereof with optimized properties compared to a specific parental antibody. A collection may be synthesized comprising the V_H domain of the parental antibody or functional fragment thereof with one or more CDR regions diversified, wherein the V_L sequence of the parental antibody or functional fragment thereof is maintained. A collection may be synthesized comprising the V_L domain of the parental antibody or functional fragment thereof with one or more CDR regions diversified, wherein the V_H sequence is the same as the parental antibody or functional fragment thereof. Alternatively, one maturation collection may be synthesized that comprises both the V_H domain and V_L domain (either in the same or separate nucleic acid molecule) of the parental antibody or functional fragment thereof, wherein each variable domain comprises one or more diversified CDRs. In some embodiments it may be desirable to optimize only the V_H or the V_L of a parent antibody or functional fragment thereof, or both. Optimizing both the V_H and V_L of a parent antibody or functional fragment thereof can be provided by at least two methods, by optimizing the V_H and V_L using separate collections or by optimizing the V_H and V_L in one collection.

Another benefit of the present disclosure is that as more is learned about the structure of antibodies and the benefit of specific amino acid sequences, the knowledge can be incorporated into the design of the collection. For example, it has been disclosed that Tregitopes (Epivax) may be designed into, modified or removed from an antibody FR or CDR regions enabling the antibodies generated to be less immunogenic in hosts. In addition, Lonza has set up the Aggresolve in silico protein analysis platform, which identifies amino acid residues that affect stability. Therefore, FR or CDR residue modifications may be incorporated into the collection design and result in antibodies or functional fragments thereof that are optimized in terms of stability or immunogenicity.

For collections of the present disclosure to be synthesized, the sequences encoding the candidate antibodies or functional fragments thereof are synthesized. At least the following companies can synthesize the nucleic acids of the present disclosure: Sloning (Puchheim, Germany), Geneart (Regensburg Germany), DNA2.0 (Menlo Park, CA USA), and Genscript (Piscataway, NJ USA).

Synthetic collections, for purposes of the present disclosure, also include collections where each candidate antibodies or functional fragments thereof is generated by gene synthesis, for example, total gene synthesis, but the portion synthesized is not the full V_H or V_L. For example, it may be advantageous to maintain a CDR3 region from the parental antibody or functional fragment thereof, in order to maintain immunogen specificity; therefore, the collection of candidate antibodies or functional fragments thereof may comprise synthetic portions of variable domains, wherein the FR1-FR3 portion is synthesized and the CDR3 and/or FR4 region(s) is subsequently cloned in by standard methods. In some embodiments, the synthetic collections may comprise fully synthetic V_H and V_L variable domains, wherein the CDR3 regions are PCR amplified and subsequently joined to the synthesized V_H and V_L regions using standard cloning techniques. For example, the V_H, FRS 1-3 and CDRs 1-2 could be gene synthesized and the FR4 and CDR3 could be cloned in by Standard methods.

In some embodiments, the synthetic collections may comprise fully synthetic V_H and V_L variable domains, wherein the CDRl, 2 or 3 regions are PCR amplified and subsequently joined to the synthesized V_H and V_L regions using standard cloning techniques.

In order to incorporate the collections of synthetic nucleic acids into vectors for display, the synthesized V_H and V_L could include restriction sites at (or near or within 5 amino acids or so) of both the 5' and 3 'ends. In addition, if it is desired to maintain the sequence encoding the parental CDR3 regions then a restriction site may be synthesized into the V_H and V_L variable domain sequences 5' to the CDR3 regions prior to synthesis. This will allow easy incorporation of the parental CDR3 regions into the collection.

Therefore, the collections of the present disclosure may include fully synthesized full length V_H and V_L domains, or variable domains where only a portion of the V_H and V_L domain is synthesized.

In some embodiments, the present disclosure enables collections, wherein said candidate antibodies or functional fragments thereof comprise substantially germline framework regions homologous to the framework regions of a parental antibody or functional fragment thereof.

In this aspect, a parental antibody or functional fragment thereof specific for an immunogen is obtained. The parental antibody or functional fragment thereof s sequence is obtained. From the sequence, the parental antibody or functional fragment thereof s V_H and V_L germline framework family is determined. For example, if the parental antibody or functional fragment thereof is found to comprise V_H 1-69 and V_LKI-39 germline genes then V_H 1-69 and V_LKI-39 collections would be selected in order to identify matured candidate antibodies or functional fragments thereof of the parental antibody or functional fragment thereof.

The present disclosure enables collections of candidate antibodies or functional fragments thereof and methods of making or using such collections, wherein more than one collection is generated, wherein each collection comprises one V_H and/or V_L germline family or gene. For example, a collection may be synthesized that comprises one germline V_H and/or one germline V_L family or gene. In these aspects, one collection of candidate antibodies or functional fragments thereof could comprise framework and/or complementarity determining region germline gene sequences of one or more germline family or genes. In theory, a collection could be made for each V_H and/or V_L germline family or gene. In these aspects, when a parental antibody of functional fragment thereof is identified, the nearest germline family or gene of the V_H and/or V_L can be identified using known methods, and the specific collection comprising candidate antibodies or functional fragments thereof with the same germline family or gene of the V_H and/or V_L can be used as the maturation collection. In these aspects, "off-the-shelf collections may be utilized for any parental antibody of functional fragment thereof identifed. After the germline family of a parental antibody or functional fragment thereof is identified, the "off-the-shelf collections corresponding to the parental antibody of functional fragment thereof s V_H and V_L germline families could be selected for purposes of maturation.

In some embodiments, the present disclosure enables collections, wherein said parental antibody or a functional fragment thereof comprises non- human sequences. Therefore, collection of the present disclosure may be used to humanize non-human parental antibodies or functional fragments thereof.

In other aspects, the collections disclosed may comprise the sequences of murine parental antibodies or functional fragments thereof. For example, a murine antibody specific for an antigen may be humanized using the above collections and methods. Maturation collections may be synthesized comprising one or more nucleic acids encoding murine variable region or CDR sequences. For example, one or more murine CDR sequences may be incorporated into the collection design. In this example, the collections may comprise murine CDR3, CDR2, and/or CDRl of either the heavy or light chain variable region from a murine parental antibody. The FR regions could be selected by any method known of skill in the art.

In some embodiments the V_H of the parental antibody can be matured using one collection, then the V_L using another collection or both the V_H and V_L using one collection.

In other aspects, the present disclosure enables a method of producing a collection of candidate antibodies or functional fragments thereof, comprising synthesizing a diverse collection of candidate antibodies or functional fragments thereof, wherein said candidates comprise substantially germline framework regions homologous to the framework region of a parental antibody or a functional fragment thereof. Certain Embodiments of the present disclosure include:

1. A substantially isolated collection of at least 1 x 10³ candidates comprising synthesized nucleic acids encoding antibodies or functional fragments thereof, wherein said nucleic acids encoding the boundaries between each framework region and each complementary determining region are substantially devoid of restriction sites, and said candidates comprise nucleic acids encoding framework regions substantially identical to the framework regions of a parental antibody or a functional fragment thereof.

2. The collection according to claim 1, wherein each candidate comprises nucleic acids encoding a CDR sequence, wherein at least one CDR sequence is unique within candidates in said collection.

3. The collection according to claim 2, wherein said candidates are based on a parental antibody or a functional fragment thereof and at least one of said candidates comprises an optimized property as compared to said parental antibody or a functional fragment thereof.

4. The collection according to claim 3, wherein said optimized property is selected from the group consisting of affinity for a specific immunogen and human characteristics.

5. The collection according to claim 1, wherein said candidates further comprise nucleic acids encoding framework regions comprising amino acid modifications as compared to said parental antibody or a functional fragment thereof.

6. The collection according to claim 5, wherein said candidates comprise an optimized property selected from the group consisting of higher binding affinity for a specific immunogen, higher binding specificity for a specific immunogen, increased human characteristics, decreased immunogenicity, higher stability, including thermal and/or serum stability, higher display rates in phage, bacterial, eukaryotic, including mammalian display, high expression levels in bacterial or eukaryotic, including mammalian, cells, a low tendency for aggregation (tendency for aggregation may be identified by Aggresolve from Lonza), removal of potential T-cell epitopes (which may be identified by Epivax or Epibase), lower deviation from germline, removal of undesired amino acid residues, such as methionines or cysteines, removal of potential glycosylation sites, enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

7. The collection according to claim 2, wherein said candidates comprise nucleic acids encoding substantially germline framework regions homologous to the framework regions of said parental antibody or a functional fragment thereof.

8. The collection according to claim 7, wherein said candidates comprise nucleic acids encoding the germline framework families substantially identical to the germline framework families of said parental nucleic acid, wherein the germline framework families comprise V_H and V_L framework families.

9. The collection according to claim 7, wherein said parental antibody or_a functional fragment thereof comprises non-human sequences.

10. The collection according to claim 1, wherein said candidates comprise synthesized nucleic acids comprising restriction sites on the 5' and 3' ends.

11. The collection according to claim 10, wherein said candidates comprise synthesized nucleic acids further comprise a restriction site 5' prime of the CDR3 region.

12. A kit comprising sequence data illustrating sequences encoding a parental antibody or a functional fragment thereof; and sequence data illustrating sequences encoding said collection according to claim 1 ; wherein said sequence data are on a readable medium. 13. A method of producing a collection of at least 1 XlO³ candidate nucleic acids, comprising synthesizing a diverse collection of candidate nucleic acids encoding antibodies or functional fragments thereof, wherein said candidate nucleic acids comprise nucleic acids encoding framework regions substantially identical to the framework regions of a parental antibody or a functional fragment thereof.

14. The method according to claim 13, wherein said candidate nucleic acids comprise nucleic acids encoding V_H regions comprising diversified CDR regions and V_L regions substantially identical to said parental antibody or a functional fragment thereof.

15. The method according to claim 13, wherein said candidate nucleic acids comprise nucleic acids encoding V_L regions comprising diversified CDR regions and V_H regions substantially identical to said parental antibody or a functional fragment thereof.

16. The method according to claim 13, wherein said candidate nucleic acids comprise nucleic acids encoding V_H and V_L regions comprising diversified CDR regions

17. The method according to claim 13, further comprising screening the antibodies or functional fragments thereof encoded by the collection, wherein the property of one or more antibodies or functional fragments thereof encoded by the collection is optimized compared to the property of said parental or a functional fragment thereof.

18. The method according to claim 17, wherein the optimized property comprises binding affinity for a specific immunogen. 19. The method according to claim 17, wherein the optimized property comprises human characteristics.

20. A method of producing a collection of at least 1 XlO³ candidate nucleic acids, comprising synthesizing a diverse collection of candidate nucleic acids encoding antibodies or functional fragments thereof, wherein said candidate nucleic acids comprise a nucleic acid encoding substantially germline framework regions homologous to the framework region of a parental antibody or a functional fragment thereof.

21. A method according to claim 20, wherein said parental antibody or a functional fragment thereof comprises non-human sequences.

22. The method according to claim 20, wherein said candidates comprise nucleic acids encoding the germline framework families substantially identical to the germline framework families of said parental nucleic acid, wherein the germline framework families comprise of V_H and V_L framework families.

23. The method according to claim 22, wherein said candidates comprise nucleic acids encoding V_H regions comprising diversified CDR regions and V_L regions substantially identical as a parental antibody or a functional fragment thereof.

24. The method according to claim 20, wherein said candidates comprise nucleic acids encoding V_L regions comprising diversified CDR regions and V_H regions substantially identical to as said parental antibody or a functional fragment thereof.

25. The method according to claim 20, wherein said candidates comprise nucleic acids encoding V_H and V_L regions comprising diversified CDR regions. 26. The method according to claim 20, further comprising screening the antibodies or functional fragments thereof encoded by the collection, wherein the property of one or more antibodies or functional fragments thereof encoded by the collection is optimized compared to the property of said parental antibody or a functional fragment thereof.

27. The method according to claim 26, wherein the optimized property comprises binding affinity for a specific immunogen.

28. The method according to claim 26, wherein the optimized property comprises human characteristics.

29. A substantially isolated collection of at least 1 x 10³ candidates comprising synthesized nucleic acids encoding proteinaceous binding molecules or functional fragments thereof.

It is to be understood that the description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications within the present invention will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the invention.

Additional Embodiments of the present disclosure include:

1. A method of identifying a candidate antibody or functional fragment thereof having an optimized property compared to a parental antibody or functional fragment thereof, comprising the steps of:

(a) identifying a parental antibody or functional fragment thereof specific for an immunogen;

(b) identifying the nucleic acid sequence encoding the parental antibody or functional fragment thereof;

(c) generating a collection of candidate antibodies or functional fragments thereof, wherein said candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions;

(d) synthesizing the collection of candidate antibodies or functional fragments thereof;

(e) screening the candidate antibodies or functional fragments thereof against said immunogen; and

(f) identifying one or more candidate antibodies or functional fragments thereof comprising an optimized property, wherein said property is optimized compared to the property of the parental antibody or functional fragment thereof.

2. The method according to claim 1, wherein said candidate antibodies or functional fragments thereof comprise framework regions based on or derived from the framework regions of the parental antibody or functional fragment thereof.

3. The method according to claims 1 or 2, wherein said candidate antibodies or functional fragments thereof comprise framework regions identical to the framework regions of the parental antibody or functional fragment thereof.

4. The method according any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise one or more complementarity determining regions based on or derived from the complementarity determining regions of the parental antibody or functional fragment thereof. 5. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline gene to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof.

6. The method according to any one of the preceding claims, wherein said property is selected from the group consisting of higher binding affinity for a specific immunogen, higher binding specificity for a specific immunogen, increased human characteristics, decreased immunogenicity, higher stability, including thermal and/or serum stability, higher display rates in phage, bacterial, eukaryotic, including mammalian display, high expression levels in bacterial or eukaryotic, including mammalian, cells, a low tendency for aggregation, removal of potential T-cell epitopes, lower deviation from germline, removal of undesired amino acid residues, such as methionines or cysteines, removal of potential glycosylation sites, and enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

7. The method according to any one of the preceding claims, wherein said parental antibody or functional fragment thereof comprises non-human sequences.

8. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise V_H regions comprising one or more diversified CDR regions and V_L regions based on or derived from said parental antibody or functional fragment thereof.

9. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified CDR regions and V_H regions based on or derived from said parental antibody or functional fragment thereof. 10. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising one or more diversified CDR regions.

11. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites.

12. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise restriction sites at the 5' and 3' ends.

13. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise a restriction site at the 5' end of the CDR3 region.

14. An isolated collection of candidate antibodies or functional fragments thereof comprising variable regions, wherein the nucleic acids encoding the variable regions are substantially devoid of restriction sites, wherein said candidate antibodies or functional fragments thereof comprise one or more diversified complementarity determining regions, and wherein one or more of said candidate antibodies or functional fragments thereof comprises an optimized property, wherein said property is optimized compared to the property of a parental antibody or functional fragment thereof.

15. The collection according to claim 14, wherein said candidate antibodies or functional fragments thereof comprise framework regions based on or derived from the framework regions of a parental antibody or functional fragment thereof. 16. The collection according to claims 14 or 15, wherein said candidate antibodies or functional fragments thereof comprise framework regions identical to the framework regions of the parental antibody or functional fragment thereof.

17. The collection according to any one of claims 14-16, wherein said candidate antibodies or functional fragments thereof comprise one or more complementarity determining regions based on or derived from the complementarity determining regions of the parental antibody or functional fragment thereof.

18. The collection according to any one of claims 14-17, wherein said candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline gene to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof.

19. The collection according to any one of the claims 14-18, wherein said property is selected from the group consisting of higher binding affinity for a specific immunogen, higher binding specificity for a specific immunogen, increased human characteristics, decreased immunogenicity, higher stability, including thermal and/or serum stability, higher display rates in phage, bacterial, eukaryotic, including mammalian display, high expression levels in bacterial or eukaryotic, including mammalian, cells, a low tendency for aggregation, removal of potential T-cell epitopes, lower deviation from germline, removal of undesired amino acid residues, such as methionines or cysteines, removal of potential glycosylation sites, and enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

20. The collection according to any one of claims 14-19, wherein said parental antibody or functional fragment thereof comprises non- human sequences. 21. The collection according to any one of claims 14-20, wherein said candidate antibodies or functional fragments thereof comprise V_H regions comprising one or more diversified CDR regions and V_L regions based on or derived from said parental antibody or functional fragment thereof.

22. The collection according to any one of claims 14-21 , wherein said candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified CDR regions and V_H regions based on or derived from said parental antibody or functional fragment thereof.

23. The collection according to any one of claims 14-22, wherein said candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising one or more diversified CDR regions.

24. The collection according to any one of claims 14-23, wherein said candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise restriction sites at the 5' and 3' ends.

25. The collection according to any one of claims 14-24, wherein said candidate antibodies or functional fragments thereof comprise variable regions, wherein the nucleic acids encoding the variable regions comprise a restriction site at the 5' prime end of the CDR3 region.

26. A kit, comprising sequence data illustrating sequences encoding a parental antibody or a functional fragment thereof; and sequence data illustrating sequences encoding said collection according to claim 14; wherein said sequence data are on a readable medium. Examples

Example 1 : Identification of a parental

A parental antibody or functional fragment thereof can be identified by any method to one of skill in the art. For example, a phage display library could be selected against an immunogen of interest. The output of the selection could be narrowed down to a number of antibodies having desired properties, such as ELISA positive. The parental antibody(ies), however, have properties, for example, affinity for a specific immunogen, that would preferably be improved. Additionally, a known, human, humanized, chimeric or murine antibody could be selected for subsequent optimization.

Example 1.2: Identify the DNA sequence of the parental

Once a parental antibody or functional fragment thereof is identified, then its nucleic acid sequence will be identified, as it is either known, or the DNA can be sequenced by known technologies.

Example 1.3: Generation of an in silico collection of candidates

The nucleic acid sequence of the parental antibody or functional fragment thereof is analyzed and a collection of candidate antibodies or functional fragments thereof is designed, generally, in silico. The parental antibody or functional fragment thereof sequence can be analyzed for the following characteristics: a) areas that may increase the risk of immunogenicity, including, but not limited to, the presence of non- human sequences, presence of potential T-cell epitopes, deviations from germline; b) a tendency for aggregation; c) undesired amino acid residues, such as methionines or cysteines; or d) potential N- linked glycosylation sites. The design of the collection of candidate antibodies or functional fragments thereof could incorporate modifications within the framework regions and/or complementarity determining regions which remove or reduce the risk of such characteristics. In addition, the design of the collection of candidate antibodies or functional fragments thereof could incorporate modifications within the framework regions and/or complementarity determining regions, which may result in optimized property including, but not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

In addition, one or more complementarity determining regions can be diversified in order to identify a candidate antibodies or functional fragments thereof having an optimized property.

Example 1.4: Synthesize in vitro

The collection of candidate antibodies or functional fragments thereof or collection of DNA encoding candidate antibodies or functional fragments thereof is generated using gene synthesis.

Example 1.5: Screening and evaluation of the candidates

The collection of candidate antibodies or functional fragments thereof is then selected against the immunogen of interest. The properties of the resulting candidate antibodies or functional fragments thereof are evaluated, and one or more candidate antibodies or functional fragments thereof are identified having an optimized property compared to the properties of the parental antibody or functional fragment thereof. The methods described below can be used to evaluate the properties of the candidate antibodies or functional fragments thereof.

Detection of improvement in affinity

In order to identify proteinaceous binding molecules, for example, antibodies, with improved affinities compared to a parental proteinaceous binding molecule, for example, antibody, the matured candidate antibodies or functional fragments thereof may be screened by any method known in the art. For example, screening by phage display selection and applying conditions of increased stringencies may be used to identify antibodies or functional fragments thereof comprising optimized affinity. The identification of the antibodies with the lowest dissociation constants, Kd, Ka, Koff may be achieved by ranking in Biacore (Schier et al. 1996; Chowdhury and Pastan, 1999; Boder et al, 2000; Yang et al, 1995). An alternative method is, e.g., affinity ranking using the BioVeris or Mesoscale Sector Imager device where Fabs obtained from crude bacterial extracts can be ranked successfully according to their affinity constants.

In some aspects, the CDRs desired to be diversified are selected by methods known to one of skill in the art. In one example, moderate improvement of affinity comprising factors of 10- to 50-fold, may be obtained by optimizing LCDR3 and HCDR2 (Yang et al, 1995; Schier et al, 1996; Chen et al, 1999).

If greater improvement factors need to be achieved, a sequential optimization of CDRs may be advisable (Yang et al, 1995; Schier et al, 1996). For example, starting with an LCDR3 optimization followed by the HCDR2 optimization, or alternatively, the HCDR2 maturation may be followed by LCDR3. If still necessary, LCDRl maturation could be done.

During affinity maturations, in most cases, it will not be sufficient to select simply for antibodies binding the target antigen. The panning parameters and stringencies preferably are designed such that the antibodies with the highest affinities to the target are predominantly enriched. For a review discussing various antibody selection methods see Bradbury and Marks (2004).

However, also antigen immobilized on solid phases was used successfully for affinity maturations (Nagy et al., 2002) and also in other groups (Gram et al., 1992; Yang et al., 1995; Chowdhury and Pastan, 1999).

Detection of improvement in binding specificity for a specific immunogen

Detection of improvement of binding specificity of a immunogen could be done by screening on different related and unrelated targets in ELISA or a Mesoscale Sector Imager device or even on protein chips.

Detection of increased human characteristics

Detection of increased human characteristics can be done by alignment with and sequence comparison of both, the initial non- human antibody and the antibody optimized for human characteristics to (1) human germline sequences and (2) rearranged antibody sequences from various databases. Detection of decreased immunogenicity

Detection of decreased immunogenicity can be done by (1) in silico analysis using T- cell epitope prediction tools such as Epibase or iTOPE or (2) using ex vivo T cell assays which determines helper CD4+ T cell responses such as Epibase IV or Episcreen. In addition, utilizing germline sequences is believed to decrease the risk of immunogenicity in humans.

Detection of increased thermal stability

Methods known to one of skill in the art could be used to identify candidate antibodies or functional fragments thereof having optimized thermal stability compared to the parental antibody or functional fragment thereof. For example, antibodies or antibody fragments can be exposed to different temperatures (4°C (on ice), 60⁰C, 70⁰C, 80°C)and then detection antibodies can be applied in order to detect the signal of properly folded antibodies. Thermal stability can also be determined by fluorescent detection of protein unfolding (Thermo fluor).

Detection of increased serum stability

Methods known to one of skill in the art would be used to identify candidate antibodies or functional fragments thereof having optimized thermal stability compared to the parental antibody or functional fragment thereof. For example, antibodies or antibody fragments can be incubated in human, mouse, rat or other serum at 37 ⁰C for a prolonged period and detection antibodies can be applied in order to detect the signal of properly folded antibodies.

Detection of an increase in display rates in phage or mammalian systems

Methods known to one of skill in the art could be used to identify candidate antibodies or functional fragments thereof having optimized display rates compared to the parental antibody or functional fragment thereof. For example, display can be evaluated by phage ELISA using capture antibodies specific for phage, such as, the anti-M13 antibody which captures phage particles via the major coat protein g8p; therefore, phage titer can be determined and an anti-Fd antibody, which binds to the displayed Fab; therefore, phages displaying Fabs can be measured. In mammalian, bacterial or yeast systems display on the cell surface can be determined by flow cytometry. Additionally, the absolute display rate can be determined by Western Blot by immobilizing Fab-loaded phage preparations onto a membrane and detecting pill fusion products with a pill specific antibody.

Detection of expression yield in bacterial or eukaryotic cells

Methods known to one of skill in the art could be used to identify candidate antibodies or functional fragments thereof having optimized expression yields compared to the parental antibody or functional fragment thereof. This can be done by expression of the parental and optimized candidates and (1) quantifying the final yield for example in an ELISA set-up directly from bacterial lysate, E. coli periplasm or cell culture supernatant, or (2) after a purification step by ultraviolet absorbance or a Bradford assay.

Evaluation of tendency for aggregation

Methods known to one of skill in the art could be used to evaluate a parental antibody or functional fragment thereof s tendency for aggregation, so that certain amino acid residues or motifs can be modified, inserted or removed in order to increase the likelihood that the candidate antibodies are less likely to aggregate. For example, a tendency for aggregation may be evaluated by the proprietary software Aggresolve from Lonza. Aggregation propensity can also be determined after expression and purification of an antibody and subsequent analysis of the monomeric and higher molecular weight fractions in analytical size exclusion chromatography.

Detection of potential T-cell epitopes

Methods known to one of skill in the art could be used to identify potential T-cell epitopes present in a parental antibody or functional fragment thereof, so that certain amino acid residues or motifs can be modified, inserted or removed in order to prevent the existence of T-cell epitopes in the candidate antibodies or functional fragments thereof.

Detection of deviation from germline

Deviations from germline can be determined by comparison to germline antibody antibody or functional fragment thereof sequences as available in IMGT, IgBLAST or vbase. The sequence of the parental antibody or functional fragment thereof and candidate antibodies or functional fragments thereof may be aligned to the germline sequences using, for example, VNTI Align X or Sequencher, in order to identify deviations from the germline sequence.

Detection of undesired amino acid residues, such as methionines or cysteines

Undesired amino acids can be detected by (1) sequence analysis to check for unpaired Cysteines or rare Methionines in the CDR regions, or (2) by correlating non-optimal biophysical characteristics with specific amino acids in CDRs or framework regions, e.g. by analysis of structure-sequence relationships, point mutagenesis etc.

Detection of potential N-linked glycosylation sites.

N-linked glycosylation sites are detectable by the consensus amino acid motifs NXS or NXT, whereby X is any amino acid except for P.

Example 2: Identifying of an peptide with improved properties

IL-6 peptide is currently being developed in the treatment of multiple inflammatory disorders. The interaction partner of IL-6 is IL-6R (CD 126).

Example 2.1 : Identify the DNA sequence of the parental

The sequence of IL-6 is known or publically available at Information Hyperlinked Over Proteins (IHOP).

Example 2.2: Generation of an in silico collection of candidates

The nucleic acid sequence of IL-6 is analyzed and a collection of candidates is designed, generally, in silico. The parental sequence can be analyzed for the following characteristics: a) areas that may increase the risk of immunogenicity, including, but not limited to, the presence of non- human sequences, presence of potential T-cell epitopes, deviations from germline; b) a tendency for aggregation; c) undesired amino acid residues, such as methionines or cysteines; or d) potential N-linked glycosylation sites. The design of the collection of candidates could incorporate modifications which remove or reduce the risk of such characteristics. In addition, the design of the collection of candidates could incorporate modifications, which may result in optimized property including, but not limited to, higher binding affinity for a specific immunogen; higher binding specificity for a specific immunogen; increased human characteristics; decreased immunogenicity; higher stability, including thermal and/or serum stability; higher display rates in phage, bacterial, eukaryotic, including mammalian, display: high expression levels in bacterial or eukaryotic, including mammalian, cells; a low tendency for aggregation; removal of potential T-cell epitopes; lower deviation from germline; removal of undesired amino acid residues, such as methionines or cysteines; removal of potential glycosylation sites; enhanced cross reactivity, including species cross reactivity, ability to bind to additional epitopes of the same target, or ability to bind different but related targets.

Example 2.3: Synthesize in vitro

The collection of candidates or collection of DNA encoding candidates is generated using gene synthesis.

Example 2.4: Screening and evaluation of the candidates

The collection of candidates is then selected against the interaction partner, IL-6R. The properties of the resulting candidates are evaluated, and one or more candidates are identified having an optimized property compared to the properties of the parental. The methods described below can be used to evaluate the properties of the candidates.

Detection of improvement in affinity

In order to identify proteinaceous binding molecules or functional fragments thereof, for example, antibodies, with improved affinities compared to a parental proteinaceous binding molecule, for example, antibody, the matured candidates may be screened by any method known in the art. For example, screening by phage display selection and applying conditions of increased stringencies may be used to identify antibodies or functional fragments thereof comprising optimized affinity. The identification of the antibodies with the lowest dissociation constants, Kd, Ka, Koff may be achieved by ranking in Biacore (Schier et al. 1996; Chowdhury and Pastan, 1999; Boder et ah, 2000; Yang et ah, 1995). An alternative method is, e.g., affinity ranking using the BioVeris or Mesoscale Sector Imager device where Fabs obtained from crude bacterial extracts can be ranked successfully according to their affinity constants.

However, also antigen immobilized on solid phases was used successfully for affinity maturations (Nagy et al, 2002) and also in other groups (Gram et al, 1992; Yang et al., 1995; Chowdhury and Pastan, 1999).

Detection of improvement in binding specificity for a specific immunogen

Detection of increased human characteristics

Detection of increased thermal stability

Methods known to one of skill in the art could be used to identify candidates having optimized thermal stability compared to the parental. For example, antibodies or antibody fragments can be exposed to different temperatures (4°C (on ice), 60⁰C, 70⁰C, 80°C)and then detection antibodies can be applied in order to detect the signal of properly folded antibodies. Thermal stability can also be determined by fluorescent detection of protein unfolding (Thermo fluor).

Detection of increased serum stability

Methods known to one of skill in the art would be used to identify candidates having optimized thermal stability compared to the parental. For example, antibodies or antibody fragments can be incubated in human, mouse, rat or other serum at 37 ⁰C for a prolonged period and detection antibodies can be applied in order to detect the signal of properly folded antibodies.

Detection of an increase in display rates in phage or mammalian systems

Methods known to one of skill in the art could be used to identify candidates having optimized display rates compared to the parental. For example, display can be evaluated by phage ELISA using capture antibodies specific for phage, such as, the anti-M13 antibody which captures phage particles via the major coat protein g8p; therefore, phage titer can be determined and an anti-Fd antibody, which binds to the displayed Fab; therefore, phages displaying Fabs can be measured. In mammalian, bacterial or yeast systems display on the cell surface can be determined by flow cytometry. Additionally, the absolute display rate can be determined by Western Blot by immobilizing Fab-loaded phage preparations onto a membrane and detecting pill fusion products with a pill specific antibody. Detection of expression yield in bacterial or eukaryotic cells

Methods known to one of skill in the art could be used to identify candidates having optimized expression yields compared to the parental. This can be done by expression of the parental and optimized candidates and (1) quantifying the final yield for example in an ELISA set-up directly from bacterial lysate, E. coli periplasm or cell culture supernatant, or (2) after a purification step by ultraviolet absorbance or a Bradford assay.

Evaluation of tendency for aggregation

Methods known to one of skill in the art could be used to evaluate a parental antibodies tendency for aggregation, so that certain amino acid residues or motifs can be modified, inserted or removed in order to increase the likelihood that the candidate antibodies are less likely to aggregate. For example, a tendency for aggregation may be evaluated by the proprietary software Aggresolve from Lonza. Aggregation propensity can also be determined after expression and purification of an antibody and subsequent analysis of the monomeric and higher molecular weight fractions in analytical size exclusion chromatography.

Detection of potential T-cell epitopes

Methods known to one of skill in the art could be used to identify potential T-cell epitopes present in a parental antibody, so that certain amino acid residues or motifs can be modified, inserted or removed in order to prevent the existence of T-cell epitopes in the candidate antibodies.

Detection of deviation from germline

Deviations from germline can be determined by comparison to germline antibody sequences as available in IMGT, IgBLAST or vbase. The sequence of the parental and candidates may be aligned to the germline sequences using, for example, VNTI Align X or Sequencher, in order to identify deviations from the germline sequence. Detection of undesired amino acid residues, such as methionines or cysteines

Detection of potential N-linked glycosylation sites.

The overall procedure is shown in Figure 1.

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Claims

ClaimsWe claim:

4. The method according any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise one or more complementarity determining regions based on or derived from the complementarity determining regions of the parental antibody or functional fragment thereof.

5. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise germline framework regions and/or complementarity determining regions, wherein said germline framework regions and/or complementarity determining regions comprise the nearest germline gene to the framework regions and/or complementarity determining regions of the parental antibody or functional fragment thereof.

9. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified CDR regions and V_H regions based on or derived from said parental antibody or functional fragment thereof.

10. The method according to any one of the preceding claims, wherein said candidate antibodies or functional fragments thereof comprise V_H and V_L regions comprising one or more diversified CDR regions.

15. The collection according to claim 14, wherein said candidate antibodies or functional fragments thereof comprise framework regions based on or derived from the framework regions of a parental antibody or functional fragment thereof.

16. The collection according to claims 14 or 15, wherein said candidate antibodies or functional fragments thereof comprise framework regions identical to the framework regions of the parental antibody or functional fragment thereof.

20. The collection according to any one of claims 14-19, wherein said parental antibody or functional fragment thereof comprises non- human sequences.

21. The collection according to any one of claims 14-20, wherein said candidate antibodies or functional fragments thereof comprise V_H regions comprising one or more diversified CDR regions and V_L regions based on or derived from said parental antibody or functional fragment thereof.

22. The collection according to any one of claims 14-21, wherein said candidate antibodies or functional fragments thereof comprise V_L regions comprising one or more diversified CDR regions and V_H regions based on or derived from said parental antibody or functional fragment thereof.

26. A kit, comprising sequence data illustrating sequences encoding a parental antibody or a functional fragment thereof; and sequence data illustrating sequences encoding said collection according to claim 14; wherein said sequence data are on a readable medium.