WO2017210149A1

WO2017210149A1 - Compositions and methods for generating an antibody library

Info

Publication number: WO2017210149A1
Application number: PCT/US2017/034918
Authority: WO
Inventors: Lior Zimmerman; Dror BARAN
Original assignee: Igc Bio, Inc.
Priority date: 2016-05-31
Filing date: 2017-05-30
Publication date: 2017-12-07

Abstract

The invention relates to compositions and methods for generating an antibody library. Specifically, the invention relates to the restriction ligation of an oligonucleotide having a complementarity determining region (CDR) heavy chain H3 (CDRH3) and a CDR light chain L3 (CDRL3) to form a full length antibody or its fragment on a plasmid, in order to generate an antibody library.

Description

COMPOSITIONS AND METHODS FOR GENERATING AN ANTIBODY LIBRARY

FIELD OF THE INVENTION

[0001] The invention relates to compositions and methods for generating an antibody library. Specifically, the invention relates to the restriction ligation of an oligonucleotide having a complementarity determining region (CDR) heavy chain H3 (CDRH3) and a CDR light chain L3 (CDRL3) to form a full length antibody or its fragment on a plasmid, in order to generate an antibody library.

BACKGROUND OF THE INVENTION

[0002] Monoclonal antibodies have been functioning as therapeutic, diagnostic and research agents since the 1970s. One of the major advancements of the last years, is the ability to develop and screen large antibody libraries for a specific target. This development is a direct consequence of phage display - a technology that enables the display of billions of proteins on top of the viral capsule. The phage display technology was followed by more technologies such as yeast display and ribosome display. [0003] Previous antibody libraries were developed by amplifying human B cells or synthesizing a completely artificial library. Antibodies cloned from B cells may not represent the full diversity of the immune system and also may have a bias towards a certain clone of sequences. Synthetic libraries may produce immunogenic antibodies that can potentially trigger an immune response in patients. [0004] Some libraries were constructed with human sequences with all or some variable (V), diversity (D), and Junction (J) (VDJ) combinations. Although the sequences of these antibodies are human, they weren't optimized for stability or developability and may raise problems upon reaching the clinical setting. The later such problems are recognized in the process, the more costly it becomes. [0005] Therapeutic antibodies must fulfill a high standard with regard to their developability, stability, immunogenicity, and functional activity. Previous generation antibody libraries, although large in number, couldn't accurately account for the vast majority of molecules in terms of stability and developability. These qualities were only determined once the antibody was screened and tested. Given that sorting methods (e.g. flow-cytometry or phage display) are known to be bound by approximately 10⁷ (flow cytometry) to 10¹¹ (phage display) variants, a reliable antibody library should be optimized in a way to maximize that every construct is developable and non-immunogenic, as well as be optimized for stability and binding specificity, to lower the probability of failure in later stages.

[0006] In order to create an antibody library in which these properties are optimized, the creators usually employ a design procedure that include several guidelines or rationale.

[0007] Encoding these guidelines in the library may result in a very diverse collection of antibodies due to the vast size of sequence/structure space of this class of molecules. A large amount of different antibodies poses a great difficulty for synthesis, as most synthesis technologies today are unable to support synthesis of large number of DNA oligos which are more than few dozens of base-pairs.

[0008] CDRH3 and CDRL3 are the major contributors to binding in an antibody molecule. Theses segments are heavily involved in binding and encode most of the antibody specificity. Furthermore, CDRH3 and CDRL3 are frequently positioned in close proximity and interact with each other. A change to either of these sequences may affect binding and/or stability. Therefore, designing and synthesizing these segments in conjunction is usually required to produce specific and developable antibodies.

[0009] Accordingly, there exists a need for an improved system and method for generating an antibody library.

SUMMARY OF THE INVENTION

[00010] In one aspect, the invention provides a recombinant nucleic acid sequence comprising: a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 by a sequence comprising one or more restriction enzyme cleavage sites.

[00011] In another aspect, the invention provides an oligonucleotide comprising: a recombinant nucleic acid sequence comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 by a sequence comprising one or more restriction enzyme cleavage sites.

[00012] In yet another aspect, the invention provides a method for generating an antibody library, the method comprising: providing an oligonucleotide, said oligonucleotide comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 via one or more restriction enzyme cleavage sites between said CDRH3 and said CDRL3; providing a vector comprising a sequence of an antibody framework comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain HI (CDRH1), a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H2 (CDRH2), and one or more sites for ligating with said first oligonucleotide having said CDRH3 and said CDRL3; facilitating the cloning of said first oligonucleotide into said vector by ligation reaction.

[00013] In some embodiments, the method may further comprise the steps of providing a second oligonulecotide, said second oligonulecotide comprising a ligation site for CDRH3 , a nucleic acid sequence of CDRL1, a nucleic acid sequence of CDRL2, and a ligation site for CDRL3, wherein said CDRH3 ligation site and CDRL1 is operably linked by a linker; cleaving said cloned vector at said one or more restriction enzyme cleavage sites between said CDRH3 and said CDRL3 ; and facilitating the cloning of said second oligonucleotide into said cleaved vector by ligation reaction, thereby obtaining a library of vectors, each having a full length of an antibody or a single chain variable fragment (scFv).

[00014] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[00015] The invention will be better understood from a reading of the following detailed description taken in conjunction with the drawings in which like reference designators are used to designate like elements, and in which: [00016] Figures 1A and IB show a schematic detailing the process of the restriction and ligation of the CDRH3-CDRL3 DNA library oligos to their compatible scFV-based plasmids, yielding full length scFV library plasmids. Figure 1A shows CDRH3-CDRL3 oligonucleotides, CDRH3-CDRL3 compatible plasmids, reaction 1 of restriction and ligation, and reaction 1 product. Figure IB shows linker oligonucleotides, CDRH3-CDRL3 plasmids, reaction 2 of restriction and ligation, and reaction 2 product.

DETAILED DESCRIPTION OF THE INVENTION

[00017] The invention provides compositions and methods for generating an antibody library. Specifically, the invention provides the restriction ligation of an oligonucleotide having a complementarity determining region (CDR) heavy chain H3 (CDRH3) and a CDR light chain L3 (CDRL3) to form a full length antibody or its fragment on a plasmid, in order to generate an antibody library.

[00018] The inventors of this application have developed a method of cloning of a rationally designed antibody library, constructed of CDRH3s and CDRL3s that are ligated together to form a short oligo that can be synthesized on a DNA chip. In a following step, restriction enzymes are used to restrict the CDRH3-CDRL3 DNA oligos so that the appropriate variable framework DNA sequences can be cloned by a 2-way ligation resulting in a full length scFV on a display or expression plasmid.

[00019] In one aspect, provided herein is a recombinant nucleic acid sequence comprising: a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 by a sequence comprising one or more restriction enzyme cleavage sites.

[00020] CDRH3 may be operably linked to one or more restriction enzyme cleavage sites, for example, at its 3' or 5' end. In one embodiment, CDRH3 may be operably linked to a restriction enzyme cleavage site at its 3' end. In another, embodiment, CDRH3 may be operably linked to a ligation site at its 5 ' end.

[00021] CDRL3 may also be operably linked to one or more restriction enzyme cleavage sites, for example, at its 3 ' or 5' end. In one embodiment, CDRL3 may be operably linked to a restriction enzyme cleavage site at its 5' end. In another, embodiment, CDRL3 may be operably linked to a ligation site at its 3 ' end.

[00022] The restriction enzyme cleavage sites of the invention may facilitate for cloning into a nucleic sequence of an antibody framework in order to result in a full length of an antibody or a single chain variable fragment (scFv) on an expression vector. [00023] Any suitable restriction enzyme cleavage site known to one of skilled in the art can be used. Examples of restriction enzyme for cleavage include, for example, but not limited to, type I, II, III, IV, V, and artificial restriction enzymes.

[00024] Type I enzymes are complex, multisubunit, combination restriction-and- modification enzymes that cut DNA at random far from their recognition sequences. Type II enzymes cut DNA at defined positions close to or within their recognition sequences. One of the most common Type II enzymes, referred to as 'Type IIS" are those that cleave outside of their recognition sequence to one side.

[00025] Type III enzymes cleave outside of their recognition sequences and require two sequences in opposite orientations within the same DNA molecule to accomplish cleavage. Type IV enzymes recognize modified, typically methylated DNA. Type V restriction enzymes utilize guide RNAs to target specific non-palindromic sequences found on invading organisms. Atrcificial restriction enzymes are well known in the art. Artificial restriction enzymes can be engineered to bind to desired DNA sequences.

[00026] In a particular embodiment, the restriction enzyme of the invention is a type II restriction enzyme, for example, a type II si endo nuclease known to one of skilled in the art.

[00027] Restriction sites and enzymes for yeast plasmid vectors are well known in the art. See e.g. , Ma et al , 1987, Gene, vol. 58, pages 201-216. Examples of restriction enzymes include, for example, but not limited to, Aatl, Aval, BamHI, Ball, BstEI, BssHII, Bsml, BSPMl, BstXl, Clal, Drain, Spel, Sphl, Bglll, Hindlll, Nrul, Kpnl, Bell, Smal, MstW, Ncol, Ndel, Nhel, Xhol, Pstl, Pvull, Hpal, EcoRl, Sail, Seal, SnaBI, Sspl, Stul, Styl, Tthl, Pvul, BspMR, Avalll, Xbal, Apal, and Xmalll.

[00028] Methods for performing a ligation reaction or assay are well known in the art. In one embodiment, a ligation reaction is performed by a ligase or an enzyme that facilitates joining of nucleic acid strands. In another embodiment, a ligation reaction is performed by a homologous recombination.

[00029] The invention also includes recombinant vectors comprising any of the nucleic acid molecules described herein. The vector may comprise a nucleic acid encoding only one antibody chain or a portion thereof (e.g. , the heavy or light chain) or a nucleic acid encoding both antibody chains or portions thereof.

[00030] Vector can be any suitable vector known to one of skilled in the art. In a particular embodiment, the vector is a plasmid vector. The term "plasmid," as used herein may refer to a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a suitable host.

[00031] In one example, the plasmid is a yeast plasmid. Yeast is organism that naturally harbour plasmids. Both circular and linier plasmids are encompassed within the scope of the invention. In one embodiment, the plasmid is a Yeast integrative plasmid (Yip). In some embodiments, Yip is a vector that relies on integration into the host chromosome for survival and replication. Yip may also be associated with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C).

[00032] In another embodiment, the plasmid is a Yeast Replicative Plasmid (YRp). In some embodiments, YRp transports a sequence of chromosomal DNA that includes an origin of replication. [00033] Other suitable vecotors are also within the scope of the invention. Other exemplary vectors include, but not limited to, phagemids, cosmids, viruses and phage nucleic acids or other nucleic acid molecules that are capable of replication in a prokaryotic or eukaryotic host. [00034] The vectors typically contain a marker to provide a phenotypic trait for selection of transformed hosts. In one example, any suitable selectable marker, known to one of skilled in the art can be used. The term "selectable marker," as used herein, may refer to a gene introduced into a cell that confers a trait suitable for artificial selection. In one example, the selectable marker is a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign nucleic acid into a cell.

[00035] In one embodiment, the selectable marker is a positive selectable marker. Positive selection marker may confer selective advantage to the host organism. In one example, the positive seletable marker is an antibiotic resistant gene, which allows the host organism to survive antibiotic selection. The host cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those colonies that can grow have successfully taken up and expressed the introduced genetic material. In one embosiment, the positve seletable marker may confer resistance to antibiotics such as, for example, ampicillin, neomycin chloroamphenicol, tetracycline, or kanamycin.

[00036] In another embodiment, the selectable marker is a negative selectable marker. Negative or counterselectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example of a negative selectable marker includes thymidine kinase, which makes the host sensitive to ganciclovir selection. [00037] In yet another embodiment, the selectable marker is a combination of positive and negative selectable marker. Such marker can serve as both a positive and a negative marker by conferring an advantage to the host under one condition, but inhibits growth under a different condition. In one example, the combination of positive and negative selectable marker includes an enzyme that can complement an auxotrophy (positive selection) and be able to convert a chemical to a toxic compound (negative selection).

[00038] Additional examples of selectable markers include, but not limited to, beta- lactamase which confers ampicillin resistance to bacterial hosts; neo gene from Tn5, which confers resistance to kanamycin and geneticin; and mutant Fabl gene (mFabl), which confers triclosan resistance to the host. [00039] In a particular embodiment, the selectable marker is a yeast marker, for example, URA3, an orotidine-5' phosphate decarboxylase from yeast, which is a positive and negative selectable marker.

[00040] Useful markers for other expression systems, are well known to those of skilled in the art. These and other selectable markers can be obtained from commercially available plasmids, using techniques well known in the art.

[00041] In one example, the start codon is a standard AUG (or ATG) codon, found in both prokaryotes and eukaryotes. In another example, the start codon is a non-AUG (or non- ATG) codon. Alternate start codons (non AUG) are very rare in eukaryotic genomes. However, naturally occurring non-AUG start codons have been reported for some cellular mRNAs. See Ivanov et ah, 2011, Nucleic Acids Research vol. 39 (10), pages 4220-4234.

[00042] The selectable marker or any gene of interest in the invention may also comprise a stop codon. In the genetic code, a stop codon (or termination codon) may refer to a nucleotide triplet within messenger RNA that signals a termination of translation. Examples of stop codon include, for example, but not limited to UAG, UAA, and UGA.

[00043] In one embodiment, the vector may be an expression vector, wherein the nucleic acid encoding the antibody is operably linked to an expression control sequence. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid molecules of the invention. The vectors may also contain genetic expression cassettes containing an independent terminator sequence, sequences permitting replication of the vector in both eukaryotes and prokaryotes, i.e. , shuttle vectors and selection markers for both prokaryotic and eukaryotic systems. When the vector may contain nucleic acids encoding both a heavy and light chain or portions thereof, the nucleic acid encoding the heavy chain may be under the same or a separate promoter. The separate promoters may be identical or may be different types of promoters.

[00044] Suitable promoters include constitutive promoters and inducible promoters. Representative expression control sequences/promoters include, for example, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha mating factors, the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, promoters derived from the human cytomegalovirus, metallothionine promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40.

[00045] In some embodiments, promoters useful in yeast expression systems include, for example, promoters from sequences encoding enzymes in the metabolic pathway such as alcohol dehydrogenase (ADH) (EPO Publication No. 284,044), enolase, glucokinase, glucose- 6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publication No. 329,203) promoters. In some embodiments, the expression construct comprises a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734), as well as promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publication No. 164,556). These and other promoters can be obtained from commercially available plasmids, using techniques well known in the art.

[00046] In another embodiment, transcription termination and polyadenylation sequences are also present in the expression constructs. These sequences are located 3' to the translation stop codon for the coding sequence. Transcription terminator/polyadenylation signal sequences are well known in the art.

[00047] A host of the present invention may be eukaryotic or prokaryotic. Suitable eukaryotic cells include yeast and other fungi, insect cells, plant cells, human cells, and animal cells, including mammalian cells, such as hybridoma lines, COS cells, NS0 cells and CHO cells. Suitable prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.

[00048] The terms "host cell", as used herein, may refer to a cell or population of cells into which a nucleic acid molecule or vector of the invention is introduced. "A population of host cells" refers to a group of cultured cells into which a nucleic acid molecule or vector of the present invention can be introduced and expressed. The host may contain a nucleic acid or vector encoding only one chain or portion thereof (e.g., the heavy or light chain); or it may contain a nucleic acid or vector encoding both chains or portions thereof, either an the same or separate nucleic acids and/or vectors. [00049] Nucleic acid molecules comprising nucleotide sequences of interest can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346. A number of appropriate host cells for use with the above systems are also known. For example, yeast hosts useful in the present invention include, but not limited to, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, and Spodoptera frugiperda. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK") cells, as well as others.

[00050] In another aspect, the present invention can be used in expression constructs to express a wide variety of substances. In an exemplary embodiment, the the present invention is used to express an antibody, for example, a monoclonal antibody, a polyclonal antibody, a humanized recombinant, or a fragment thereof. In one embodiment, the invention is used to express the nucleic acid sequence that encodes a heavy or light chain immunoglobulin.

[00051] The vector of the invention can be constructed by methods or techniques well known in the art. Construction of yeast strains are well known and fully disclosed, for example, in U.S. Patent 5,635,369 and U.S. Patent Application Publication 2002/0160380, which are incorporated by reference herein in their entirety. Plasmid construction by homologous recombination yeast is also known in the art. See e.g., Ma et al , 1987, Gene, vol. 58, pages 201-216, which is incorporated by reference herein in its entirety. [00052] A wide variety of methods, known to one of skilled in the art, can be used to deliver the expression constructs to cells. Such methods include, for example, but are not limited to, DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyomithine-mediated transfection, electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection.

[00053] The invention also includes methods of producing an antibody of the present invention, which entails culturing a host cell expressing one or more nucleic acid sequences encoding an antibody of the present invention, and recovering the antibody from the culture medium. In certain embodiments, the antibody is purified by separating it from the culture medium. Antibodies comprising more than one chain can be produced by expressing each chain together in the same host; or as separate chains, which are assembled before or after recovery from the culture medium.

[00054] Any suitable antibody or fragment thereof can be produced. Examples of an antibody expressed by the invention includes, but are not limited to, anti- anti-EGFR antibodies such as ERBITUX (cetuximab), ABX-EGF, IMC-C225, and Merck Mab 425; IGF- IR antibody, an antibody that inhibits erbB2 receptor, EGF-R, CD20 or VEGF; antibodies, for example, DC101, Mab h2C10 (U.S. 7,241,444), 20D7S, CS1, and CS4; anti-17-lA cell surface antigen antibodies such as PANOREX (edrecolomab); anti-4-lBB antibodies; anti- 4Dc antibodies; anti-A33 antibodies such as A33 and CDP-833; anti-.alpha.4.beta.l integrin antibodies such as natalizumab; anti-complement factor 5 (C5) antibodies such as 5 G 1.1; anti- CA125 antibodies such as OVAREX (oregovomab); anti-CD3 antibodies such as NUVION (visilizumab) and Rexomab; anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B and Oncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodies such as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7, 2H7.vl6, 2H7.vl l4, 2H7.vl l5, BEXXAR (tositumomab, 1-131 labeled anti- CD20), RITUXAN (rituximab), and ZEVALIN (Ibritumomab tiuxetan, Y-90 labeled anti- CD20); anti-CD22 antibodies such as LYMPHOCIDE (epratuzumab, Y-90 labeled anti- CD22); anti-CD23 antibodies such as IDEC-152; anti-CD25 antibodies such as basiliximab and ZENAPAX (daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30; anti-CD33 antibodies such as MYLOTARG (gemtuzumab ozogamicin), Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodies such as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8, ANTOVA, and IDEC-131; anti-CD44 antibodies such as bivatuzumab; anti-CD46 antibodies; anti-CD52 antibodies (alemtuzumab); anti-CD55 antibodies such as SC-1 ; anti-CD56 antibodies such as huN901-DMl ; anti-CD64 antibodies such as MDX-33; anti-CD66e antibodies such as XR-303; anti-CD74 antibodies such as IMMU-110; anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89 antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodies such as B-B4-DM1; anti- CD 146 antibodies such as AA-98; anti-CD148 antibodies; anti-CEA antibodies such as CT84.66, labetuzumab, and PENTACEA; anti-CTLA-4 antibodies such as MDX-101; anti- CXCR4 antibodies; anti-EpCAM antibodies such as Crucell's anti-EpCAM, ING-1, and IS- IL-2; anti-ephrin B2/EphB4 antibodies; anti-Her2 antibodies such as HERCEPTIN, MDX- 210; anti-FAP (fibroblast activation protein) antibodies such as sibrotuzumab; anti-ferritin antibodies such as NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8 antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250 antibodies such as WX- G250 and RENCAREX; anti-GD2 ganglioside antibodies such as EMD-273063 and TriGem; anti-GD3 ganglioside antibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIa antibodies such as ReoPro; anti-hep arinase antibodies; anti-HLA antibodies such as ONCOLYM, Smart 1D10; anti-HM1.24 antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptor antibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164; anti- IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such as CNTO-328 and elsilimomab; anti-IL-15 antibodies such as HUMAX-IL15; anti-KDR antibodies; anti-laminin 5 antibodies; anti- Lewis Y antigen antibodies such as Hu3S193 and IGN-311 ; anti-MCAM antibodies; anti-Mucl antibodies such as BravaRex and TriAb; anti- NC AM antibodies such as ERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn and Therex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptk antibodies; anti-PTN antibodies; anti- RANKL antibodies such as AMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as Monopharm C; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA and MDX-220; anti-TGF-.beta. antibodies such as CAT- 152; anti-TNF-. alpha, antibodies such as CDP571, CDP870, D2E7, HUMIRA (adalimumab), and REMICADE (infliximab); anti-TRAIL-Rl and TRAIL-R2 antibodies; anti-VE-cadherin-2 antibodies; anti-VLA-4 antibodies; antibodies to treat autoimmune or inflammatory disease; antibodies to treat transplant rejection; antibodies to treat infectious diseases, for example, anti-anthrax antibodies such as ABthrax, anti-CMV antibodies such as CytoGam and sevirumab, anti- cryptosporidium antibodies such as CryptoGAM, Sporidin-G, anti-helicobacter antibodies such as Pyloran, anti-hepatitis B antibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such as HRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab, RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb, BSYX-A110, and SE-Mab or their combinations.

[00055] The invention also provides a kit comprising one or more oligonucleotides of the invention, one or more vectors of the invention, one or more host cells of the invention, one or more restriction enzymes of the invention, one or more ligases or enzymes that facilitate the joining of nucleic acid strands, or a combination thereof.

[00056] Kits for contructing vectors of or performing assays are also provided by the present invention. The kits can include a container that includes some or all of the reagents and methods for contructing vectors or carrying out the assays. The kits can also include labels or instructions for contructing vectors or carrying out the assays.

[00057] The term "nucleic acid," as used herein, can include both double- and single- stranded sequences and refers to, but not limited to, cDNA from yeast, viral, procaryotic or eucaryotic mRNA, genomic DNA sequences, or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.

[00058] The term "operably linked," as used herein, refers to an arrangement of elements wherein the components so described are configured so as to perform their desired function. [00059] The term "recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[00060] The term "promoter," as used herein, refers to a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3' direction) coding sequence operably linked thereto. For purposes of the present invention, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but not always, contain "TATAA" boxes and "CAAT" boxes.

[00061] The term "host cell," as used herein, refers to a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence.

[00062] The term "expression construct," as used herein, refer to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. The expression construct includes control elements, as described above, such as a promoter or promoter/enhancer which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression construct described herein may be contained within a plasmid construct. In addition to the components of the expression construct, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single- stranded DNA (e.g., an origin of replication).

[00063] As used herein, the term "antibody" includes intact immunoglobulin molecules comprising 4 polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino acid sequences of their constant domains. The variable regions of kappa light chains are referred to herein as VK. The expression of VL, as used herein, is intended to include both the variable regions from kappa- type light chains (VK) and from lambda-type light chains. The light chain constant region is comprised of one domain, CL. The VH and VL regions include regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1- CDR1 -FR2-CDR2-FR3-CDR3-FR4. "CDRHl" refers to the first CDR region in an antibody heavy chain, "CDRH2" refers to the second CDR region in an antibody heavy chain, and "CDRH3" refers to the third CDR region in an antibody heavy chain. "CDRL1" refers to the first CDR region in an antibody light chain, "CDRL2" refers to the second CDR region in an antibody light chain, and "CDRL3" refers to the third CDR region in an antibody light chain.

[00064] The term "antibody" as used herein is also intended to encompass intact antibodies, functional fragments which bind antigen, and variants thereof which bind antigen, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof; each containing at least one CDR. Antibodies of the invention include antibody fragments or variants having one, two, three, four, five, six or more CDR regions.

[00065] Antibody fragments which are embraced by the present invention include Fab (e.g. , by papain digestion), facb (e.g. , by plasmin digestion), pFc' (e.g. , by pepsin or plasmin digestion), Fd (e.g. , by pepsin digestion, partial reduction and reaggregation), sVds, and Fv or scFv (e.g. , by molecular biology techniques). Antibody fragments are also intended to include domain deleted antibodies, diabodies, triabodies, linear antibodies, single-chain antibody molecules (including camelized antibodies), and multispecific antibodies formed from antibody fragments. [00066] The term "antibody, " as used herein, also includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g. , mouse or rat) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Thus, the present invention includes, for example, chimeric antibodies comprising a chimeric heavy chain and/or a chimeric light chain. The chimeric heavy chain may comprise any of the heavy chain variable (VH) regions described herein or mutants or variants thereof fused to a heavy chain constant region of a non-human antibody. The chimeric light chain may comprise any of the light chain variable (VL) regions described herein or mutants or variants thereof fused to a light chain constant region of a non-human antibody.

[00067] Antibodies of the invention also include "humanized antibodies", which are antibody molecules having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, or improve, antigen binding. These framework substitutions are identified standard techniques such as by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. Antibodies can be humanized using a variety of techniques including CDR- grafting, veneering or resurfacing, and chain shuffling.

[00068] The term "human antibody," as used herein, includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The human antibody can have at least one position replaced with an amino acid residue, e.g. , an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[00069] Antibodies of the invention also include "recombinant human antibody," which includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. [00070] Antibodies of the present invention can be monospecific, bispecific or multispecific. Monospecific antibodies bind to only one antigen. Bispecific antibodies (BsAbs) are antibodies that have two different antigen-binding specificities or sites. Multispecific antibodies have more than two different antigen-binding specificities or sites. Where an antibody has more than one specificity, the recognized epitopes can be associated with a single antigen or with more than one antigen.

[00071] In another aspect of the invention, the antibody is conjugated to another moiety, either directly or indirectly. The conjugation may be chemical or biosynthetic. Other moieties which can be conjugated to the antibodies include toxins, anti-tumor agents, detectable labels, target moieties and reporter moieties.

[00072] All patents, patent applications, and scientific publications cited herein are hereby incorporated by reference in their entirety.

[00073] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

EXAMPLE 1

Process for Generating an Antibody Library

[00074] We have developed a method of cloning of a rationally designed antibody libraries, constructed of CDRH3s and CDRL3s that are ligated together to form a short oligo that can be synthesized on a DNA chip. In a following step, type lis restriction enzymes are used to restrict the CDRH3-CDRL3 DNA oligos so that the appropriate V framework DNA sequences can be cloned by a 2-way ligation resulting in a full length scFV on a display or expression plasmid [00075] Figure 1 shows a schematic detailing the process of the restriction and ligation of the CDRH3-CDRL3 DNA library oligos to their compatible scFV-based plasmids, yielding full length scFV library plasmids. [00076] As shown in Figure 1, a DNA sequence composing the two CDRH3 loop and CDRL3 is joined together with two type lis restriction sites with a stuffer in between. Two additional restriction sites are added at the 3 ' and 5 ' edges of the sequence. The first restriction sites, are intended for the restriction-ligation of the VL fragment, while the second set of restriction sites are intended for the restriction ligation of the oligo to a VH containing plasmid.

[00077] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A recombinant nucleic acid sequence comprising: a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 by a sequence comprising one or more restriction enzyme cleavage sites.

2. The nucleic acid sequence of claim 1, wherein said CDRH3 is operably linked to a restriction enzyme cleavage site at its 3 ' end.

3. The nucleic acid sequence of claim 1, wherein said CDRL3 is operably linked to a restriction enzyme cleavage site at its 5 ' end.

4. The nucleic acid sequence of claim 1, wherein said CDRH3 is operably linked to a ligation site at its 5 ' end

5. The nucleic acid sequence of claim 1, wherein said CDRL3 is operably linked to a ligation site at its 3 ' end.

6. The nucleic acid sequence of claim 1, wherein said one or more restriction enzyme cleavage sites facilitate for cloning into a nucleic sequence of an antibody framework in order to result in a full length of an antibody or a fragment thereof on an expression vector.

7. The nucleic acid sequence of claim 1, wherein each of said one or more restriction enzyme cleavage sites is cleavable by a type II restriction enzyme.

8. The nucleic acid molecule of claim 7, wherein said restriction enzyme is a type lis restriction enzyme.

9. A vector comprising said recombinant nucleic acid sequence of claim 1.

10. The vector of claim 9, wherein said vector is a plasmid vector.

11. The vector of claim 10, wherein said plasmid is a self-replicating plasmid.

12. The vector of claim 10, wherein said plasmid is a yeast plasmid.

13. The vector of claim 9, further comprising a sequence encoding a selectable maker.

14. The vector of claim 13, wherein said selectable marker is a reporter gene that indicates the success of a transfection or transformation to introduce a foreign nucleic acid into a cell.

15. A library comprising a vector of claim 9.

16. A host cell comprising a vector of claim 9.

17. A method for generating an antibody library, the method comprising:

providing a first oligonucleotide, said first oligonucleotide comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 via one or more restriction enzyme cleavage sites between said CDRH3 and said CDRL3 ;

providing a first vector comprising a sequence of an antibody framework comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain HI (CDRHl), a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H2 (CDRH2), and one or more sites for ligating with said first oligonucleotide having said CDRH3 and said CDRL3;

facilitating the cloning of said first oligonucleotide into said first vector by a ligation reaction;

providing a second oligonulecotide, said second oligonulecotide comprising a ligation site for CDRH3, a nucleic acid sequence of CDRLl, a nucleic acid sequence of CDRL2, and a ligation site for CDRL3, wherein said CDRH3 ligation site and CDRLl is operably linked by a linker;

cleaving said cloned vector at said one or more restriction enzyme cleavage sites between said CDRH3 and said CDRL3 ; and facilitating the cloning of said second oligonucleotide into said cleaved vector by a ligation reaction,

thereby obtaining a library of vectors, each having a full length of an antibody or a fragment thereof.

18. The method of claim 17, wherein said CDRH3 is operably linked to a restriction enzyme cleavage site at its 3 ' end.

19. The method of claim 17, wherein said CDRL3 is operably linked to a restriction enzyme cleavage site at its 5 ' end.

20. The method of claim 17, wherein said CDRH3 is operably linked to a ligation site at its 5' end

21. The method of claim 17, wherein said CDRL3 is operably linked to a ligation site at its 3' end.

22. The method of claim 17, wherein said one or more restriction enzyme cleavage sites facilitate for cloning into a nucleic sequence of an antibody framework in order to result in a full length of an antibody or a fragment thereof on an expression vector.

23. The method of claim 17, wherein each of said one or more restriction enzyme cleavage sites is cleavable by a type II restriction enzyme.

24. The method of claim 23, wherein said restriction enzyme is a type II si restriction enzyme.

25. The method of claim 17, wherein said first oligonucleotide further comprises a sequence encoding a selectable maker.

26. The method of claim 25, wherein said selectable marker is a reporter gene that indicates the success of a transfection or transformation to introduce a foreign nucleic acid into a cell.

27. The method of claim 17, wherein said vector is a plasmid vector.

28. The method of claim 17, wherein said linker is Gly₄Ser₃ linker.

29. The method of claim 27, wherein said plasmid is a self-replicating plasmid.

30. The method of claim 27, wherein said plasmid is a yeast plasmid.

31. A library of antibodies or single chain variable fragments (scFv) produced by the method of claim 17.

32. An oligonucleotide comprising the recombinant nucleic acid sequence of claim 1.

33. A method for generating an antibody library, the method comprising:

providing an oligonucleotide, said oligonucleotide comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H3 (CDRH3) and a nucleic acid sequence of complementarity determining region (CDR) of light chain L3 (CDRL3), wherein said CDRH3 is fused to said CDRL3 via one or more restriction enzyme cleavage sites between said CDRH3 and said CDRL3 ;

providing a vector comprising a sequence of an antibody framework comprising a nucleic acid sequence of complementarity determining region (CDR) of heavy chain HI (CDRHl), a nucleic acid sequence of complementarity determining region (CDR) of heavy chain H2 (CDRH2), and one or more sites for ligating with said first oligonucleotide having said CDRH3 and said CDRL3;

facilitating the cloning of said first oligonucleotide into said vector by ligation reaction, thereby generating said antibody library.