PHAGE DISPLAY LIBRARIES OF HUMAN VH FRAGMENTS

Abstract: Phage display libraries are taught in which the recombinant phage population displays a plurality of potential binding fragments having preferred characteristics of solubility and/or intermolecular interaction. Also taught are methods of biasing display libraries to produce variants which more closely approximate the preferred characteristics of the parental binding fragment.

Description: PHAGE DISPLAY LIBRARIES OF HUMAN V H FRAGMENTS Field of the Invention The present invention relates to combinatorial libraries including phage display libraries which display single domain heavy chain binding fragments having preferred characteristics of solubility. Background of the Invention Developments in antibody engineering and recombinant DNA technology have made it possible to generate forms of recombinant antibody fragments which, in many ways, are functional substitutes of larger intact im unoglobulin molecules. Single heavy domain antibody fragments ("dAb") have been the subject of several reports in the patent and scientific literature. The literature reports efforts to generate phage display libraries of such fragments for biopanning against a target ligand. U.S. Patent No. 5,702,892 ('892) discloses a phage display library constructed in an Ml 3 derived expression vector, in which recombinant phage of the library contain a polynucleotide encoding a fusion protein which comprises a phage coat protein and an immunoglobulin heavy chain binding-fragment. The heavy-chain binding-fragment spans from a position upstream of CDR1 to a position downstream of CDR3. '892 describes that the DNA sequence encoding the CDR3 region and/or the CDR1 region may be randomly varied so that the population of phage expresses a series of potential heavy chain binding domains for panning against the target ligand. U.S. Patent No. 5,759,808 discloses a phage display library comprising a population of phage based on random variation of a cDNA sequence obtained from lymphocytes of camelids previously immunized with target antigens. Camelid heavy chain antibodies occur naturally, in a composition of about 45%, as heavy chain dimers. Heavy chain antibodies specific for a target antigen may be generated by immunizing a member of the camelid species with the target antigen (see Lauwereys et al. (1998) The EMBO J. 17, 3512-3520). Hamers-Casterman et al. (1993) Nature 363, 446-448 report that camelid heavy chain antibodies are naturally more hydrophilic at amino acid residues at locations 44, 45 and 47 (Kabat numbering system), in FR2, which corresponds to the surface where they normally contact the VL domain. Another salient feature of a camelid V H is that it generally has a comparatively longer CDR3 with a high incidence of cysteines and thus may form, via paired cysteines in CDR1 and CDR3, exposed loops, which are more amenable to binding into cavities such as the active site of enzymes and antibodies (Desmyter et al. (1996) Nat. Struct. Biol. Vol. 3, No. 9, p. 803). However, it has been questioned whether single domain antibodies with desired affinities can be generated with such configurations in the absence of prior immunization, i.e. with a naϊve library (Lauwereys et al. (1998) supra). The present invention discloses advances in the technology related to creating libraries containing immunoglobulin-like proteins that specifically bind target ligands eg. antigens. Summary of the Invention According to one aspect, the invention is directed to a library for expression of immunoglobulin heavy chain domains, said library comprising a repertoire of nucleic acid sequences each encoding a polypeptide comprising a N H , said N H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-Ν, 93-A and 108-Q, said repertoire comprising a diversity of sequences which differ from one another at least in a subsequence coding for part of the CDR so as to provide nucleic acid encoding a repertoire of polypeptides comprising V differing at least in said CDR and comprising at least one amino acid of said group. In addition to a library of nucleic acid molecules, the term expression library is understood to specifically include a phage, viral, bacterial or other cell surface display library, a ribosome display library or any other functional nucleic acid expression system which permits the expression products to be screened. According to another aspect, the invention is directed to a method for generating a V H expression library having a diversity of CDR sequences, said method comprising: providing expression vectors, said vectors comprising a nucleic acid sequence which encodes a polypeptide comprising a V H , said V H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q; introducing by mutagenesis a diversity of CDR sequences into said V H nucleic acid sequence; and recovering an expression library having a diversity of binding activities. According to another aspect, the invention is directed to an expression library which expresses polypeptides comprising Ns, said library comprising a set of framework regions carrying a diversity of CDR sequences, said library having a diversity of binding activities, said frame work regions comprising one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-Ν, 93-A and 108-Q. The invention is also directed to a method of identifying a polypeptide comprising a V ) which binds to a target ligand, the method comprising the steps of screening polypeptide members of this immediately aforementioned expression library for their respective ability to bind to the target ligand; and identifying at least one polypeptide member which binds to the target ligand. According to another aspect, the invention is directed to a polypeptide comprising a V H , said polypeptide derived from a library according to any of the preceding paragraphs. The invention is directed to a population of variants of at least one parental N H ligand- binding molecule (dAb), wherein said parental N H ligand-binding molecule comprises an immunoglobulin N H binding fragment comprising, at least in substantial part, at least the framework (FR) regions of the immunoglobulin N H fragment depicted Figure 2, wherein said variants comprise at least in substantial part, the FR regions of the immunoglobulin N H fragment depicted Figure 2, including at least one (and preferably all) of amino acid residues, 6, 23, 82a, 93, and 108, and differ from said parental ligand-binding molecule in amino acid residues constituting at least part of at least one of the CDRs of said parental ligand-binding molecule: Preferably said population of variants is constimted by one or more combinatorial libraries of such variants, for example, protein arrays, phage display libraries, ribosome display libraries etc. It is to be understood that the variants may (though not necessarily) form part of another structure or molecule, for example in the case of phage display, part of the coat protein of the phage. Accordingly, the term variant is used broadly to refer to variants of the essential molecule (a ligand-binding molecule) when forming part of another structure or molecule (eg. as in phage display or ribosome display) or when independent of any such combination, eg. in the case of protein arrays whose members may not be associated with individual supporting structures/molecules. hi a particularly preferred embodiment said parental NH ligand binding molecule and said variants comprise each of amino acid residues 6, 23, 82a, 93 and 108, depicted in Figure 2 and more preferably each of amino acid residues 6, 23, 74, 82a, 83, 84, 93, and 108 depicted in Figure 3. More preferably, said parental V H ligand binding molecules and said variants include the entirety of the FR regions the parental N H ligand molecule depicted in Figure 2 or 3, optionally subject to one or more additions, deletions and/or substitutions, (preferably conservative amino acid substitutions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants. Optionally, said parental N H ligand binding molecules and variants additionally include, the entirety of the CDR1 and CDR2 depicted in Figure 2 or 3, optionally subject to one or more additions, deletions and/or substimtions (preferably conservative amino acid substimtions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants. In a particularly preferred embodiment, said parental N H ligand molecule and said variants, additionally comprise, the entirety of CDR3 of the parental N H ligand binding molecule depicted in Figure 2 with the exception of amino acids lOOi to lOOn, which are randomized to create said population of variants subject only to one or more additions, deletions, and/or substimtions, (preferably conservative amino acid substimtions) which, if they do not improve, preferably at least do not adversely affect the solubility properties of said variants. In another embodiment, said parental N H ligand binding molecule and said variants comprise one or more amino substimtions relative to Figure 2, including any combinations thereof. hi a further preferred embodiment nucleotides coπesponding to amino acid residues 3-16 are optimized to remove a putative recombination site. For purposes herein, it is to be understood that an adverse affect on the solubility properties is to be assessed in terms of the percentage of dimer and higher aggregate forms, relative to monomer, as determined by size exclusion chromatography (i.e. the respective areas under the peaks representing monomer, dimer and higher aggregate forms, as illustrated in Figure 5). In another aspect, the invention is directed to a ligand-binding molecule or variant which has been identified as binding to a target ligand by screening a combinatorial library of the invention for one or more ligand-binding molecules which specifically recognize said target ligand. The invention is also directed more generally to any specific such ligand- binding molecule which is derived from such combinatorial library of the invention. It is understood herein that such specific ligand-binding molecule may be directly obtained from such a library or may be indirectly derived, for example, through the course of further antibody engineering or other modification steps (eg. creating fragments, derivatives, a secondary library, etc) using a ligand-binding molecule directly or indirectly obtained from such library. It also understood that the invention excludes known ligand-binding molecules. In one embodiment of this aspect of the invention the target ligand is a cancer antigen. Figures 2, 3 and 4 depict variations, more fully described below, on the preferred immunoglobulin NH ligand binding fragment and/or nucleic acid construct depicted in Figure 1. Figure 1 describes a wild-type parental immunoglobulin V H binding fragment derived from human monoclonal antibody BT32/A6 (hereinafter referred to as "A6") partially described in U.S Patent No. 5,639,863 (hereinafter referred to as the '863 patent). As described in our co-pending application PCT/CA00/01027, the contents of which are hereby incorporated by reference, A6 has preferred solubility and other characteristics which lend themselves well to the creation of libraries, including naϊve libraries, of various types of human immunoglobulin fragments including dAbs as more fully described below. Accordingly, A6, and in particular, as more fully described below, at least a substantial part of the framework (FR) regions of the A6 V H fragment depicted in Figure 2, alone or in combination with features of its CDR3, provides a useful departure point, in the form of a parental ligand-binding molecule, for the randomization or partial randomization of amino acid residues which tend to play a predominant role in ligand-binding, namely the CDR regions of the heavy chain and particularly the CDR3 of the heavy chain. As more fully described below, the nucleic acid changes (removal of the recombination site) relative to A6 wild-type (Figure 1) reflected in Figure 4 may be incorporated into Figures 2 and 3 respectively. The combinatorial library of the invention may be generated by phage display. In a preferred embodiment of one aspect of the invention, the invention is directed to a phage display library displaying a plurality of different variants of a parental V H ligand-binding molecule, wherein said parental ligand-binding molecule comprises an immunoglobulin N H binding fragment comprising, at least in substantial part, at least the FR regions of the immunoglobulin NH fragment depicted in Figure 3 including at least one of amino acid residues 6, 23, 74, 82a, 83, 84, 93 and 108, and wherein said variants are encoded by nucleic acid sequences which vary from the nucleic acid sequence encoding said parental ligand- binding molecule in a subsequence (at least one) encoding at least part of one of the CDRs of said parental ligand-binding molecule, preferably the CDR3, whereby said plurality of variants comprise at least, in substantial part, the FR regions of the immunoglobulin NH fragment depicted in Figure 3, including at least one of said amino acid residues and are differentiated, at least in part, by amino acid variations encoded by variations in said subsequence. In a preferred embodiment, in addition to substantial preservation and optional improvement of the FR regions of A6, the A6-based parental ligand molecule comprises (and therefore preserves within members of the library), in substantial part (subject to at least partial randomization of selected regions of one of the CDRs (preferably the CDR3), to create binding diversity within the library, one or more of the CDR regions of the A6 N H fragment. In a further preferred embodiment, at least the length of the wild-type N H CDR3 (23 amino acids) and preferably also elements of its amino acid composition, is preserved or at least partially preserved (approximately 16-23 amino acids and more particularly 18 to 23 amino acids). Optionally the CDR3 may also be lengthened by approximately 1 to 10 residues. The library may optionally have representation of binding molecules having CDR3s of varying lengths. It is known that a dAb molecule, due to the removal of its light chain partner, tends to, in most, if not all cases, aggregate, in varying degrees due to the "sticky" nature of the NL interface. This stickiness is attributable, at least in part, to the hydrophobic nature of the N H residues at this interface. This stickiness results in substantial dimer and/or multimer formation which may reduce, on the whole, the solubility characteristics of members of the library. Accordingly, in a further preferred aspect of the invention A6 NH amino acid residues at the NL interface are substitated by residues which tend to minimize aggregate formation, for example, hydrophilic amino acids, including one or more of the highlighted substitutions reflected in Figure 8, relative to Figure 1. Alternatively, in yet a further preferred embodiment of the invention, more fully described below, such substitutions are not fixed within the entire population of the library, but are introduced by randomizing or partially randomizing various A6 VH amino acid residues, particularly including FR residues, among the residues at the interface, (see for example, Padlan et al "Anatomy of the Antibody Molecule" Molecular Immunology Vol. 31, pl69-217, Table 25 for itemization and related discussion of these residues). Particularly in the case of larger size libraries, for example those generated by ribosome display, it is possible to introduce diversity in one or more FR regions containing amino acid residues identified herein as important for improving solubility properties, in addition to one or more regions affecting the specificity of the dAb, eg. the V H CDR3 and CDR1. Alternatively, in yet a further preferred embodiment of the invention, FR regions, other than, or in addition to, modifications to the VL interface (FR2) may be modified by at least partial randomization, for example, one or both of FRl (one or more of residues 4 to 21) and FR4 (one or more of residues lOOo to 113) to improve, on the whole, the solubility characteristics of members of the library (for example, biasing at least some and preferably all of one or both of these sets of residues [at least 70% or more], preferably 90% in favour of the parental amino acid constitution to achieve 10% randomization). In the case of A6 dAb fragments, it has been found that recombination events within the nucleic acid sequence encoding the V H binding fragment tend to result in deletions yielding shorter molecules, with possible compromise in binding characteristics. Thus, in a further preferred aspect of the invention nucleic acid sequences which promote such recombination events (at putative recombination sites) are substitated, to oppose this tendency, preferably in a manner that does not result in an amino acid change. These changes may be incorporated, for example, into the nucleotide sequences encoding the parental V H ligand-binding molecule depicted in Figure 2 or 3. In particularly preferred aspects, the present invention provides a heterogeneous population of genetic packages (eg. phage) having a genetically determined outer surface protein, wherein the genetic packages collectively display a plurality of different, preferably human (i.e. having substantial identity, preferably at least 80% homology to human framework and other conserved regions) V H ligand-binding fragments, each genetic package including a nucleic acid construct coding for a fusion protein which comprises at least a portion of the outer surface protein and a variant of at least one soluble parental ligand- binding fragment preferably derived from or having a substantial part of the FR regions of the amino acid sequence identified in one of Figures 2 or 3 (or a sequence at least 80%, preferably 85 to 100%, more preferably 90-100%, homologous (% identity) thereto), including at least one and preferably all of amino acid residues 6, 23, 82a, 93 and 108 " depicted in Figure 2 or 3, wherein the variant V H ligand-binding fragments preferably span from a position upstream of an immunoglobulin heavy chain CDR1 to a position downstream of CDR3 (preferably including substantially all of FRl and/or FR4), and wherein at least part of a CDR, preferably the CDR3, is a randomly generated variant of a CDR of said parental V H ligand binding-fragment and wherein the fusion protein is preferably expressed in the absence of an immunoglobulin light chain whereby the variant V H ligand-binding fragments are, on the whole, better adapted to be or better capable of being expressed as soluble proteins. Various combinations of 2,3 and 4 substitations relative to wild-type A6 at amino acid residues 6, 23, 82a, 93 and 108 are also contemplated by the invention. In yet another embodiment of the invention, by biasing the amino acid constitution, preferably on an individual amino acid by amino acid basis, in favor of the wild-type or parental amino acid constitution, even portions of the parental ligand-binding molecule that are randomized in favor of generating variability in the variant binding fragments can be engineered to maintain favorable solubility characteristics of the parental binding domain. In one embodiment of the invention, a portion of the construct encoding at least part of the CDR3 is biased or partially biased in favor of the parental amino acid constitution. In a further prefeπed embodiment, the parental V H binding-fragment naturally has a long CDR3 that is amenable to forming exposed loops for binding into cavities. In a most preferred embodiment, the parental V H ligand-binding fragment is built on a human framework or is adapted from or adaptable to a human framework. In another preferred embodiment, the preferred binding region of the variants (corresponding to the randomized or partially randomized part of the CDR3) is located in carboxy terminal region of the CDR3. In summary, according to a prefeπed embodiment of the invention, a substantial part of the amino acid sequence identified in Figure 2, preferably including at least part of the CDR3, supplies the preferred amino acid constitution of the various preferred parental ligand- binding molecules, such that a population of variant heavy chain ligand-binding molecules built on this framework of amino acids are on the whole better adapted to be or better capable of being expressed as soluble proteins. Generally, the importance of amino acid residues 6, 23, 82a, 93, and 108, shown in Figure 2, particularly when all five are combined, is that they can be used to significantly augment the solubility properties of a parental V H ligand binding fragment, preferably one, like A6, that has useful solubility properties to begin with, to produce a library of dAb variants for panning against a target ligand, said variants on the whole being better adopted to be or better capable of being expressed as soluble proteins. Brief Description of the Drawings The invention will now be described with reference to the drawings, wherein: Figure 1 is a sequence diagram showing the A6 V H ligand-binding molecule from which we construct parental V H ligand binding molecules according to the invention. Figure 2 is a sequence diagram showing a preferred parental V H ligand-binding molecule (A6N H -L1) according to the invention, incorporating amino acid substitations E6A, S23A, S82aΝ, 93A and T108Q. Figure 3 is a sequence diagram showing the A6V H -L2 ligand-binding molecule according to the invention, incorporating 3 additional amino acid substitations at positions 74, 83 and 84. Figure 4 is a sequence diagram showing the A6V H ligand-binding molecule (encoded by A6 chi (-)) according to the invention, in which nucleic acids corresponding to amino acids 3 to 16 of A6 have been modified to remove a putative recombination site, leaving the amino acid constitution of A6 unchanged. Figure 5 is a size exclusion chromatogram of A6V H -L1 dAb following IMAC purification comparing A6V H -L1 (plot A) to wildtype A6V H (plot B) and showing a much greater proportion of monomer (represented by the largest peak). Plot C represents a camelized version of A6 with amino acid substitutions of positions 44, 45, 47 and 94 as described herein and in the literature. Figure 6 is a size exclusion chromatogram of Yst9.1-L3-9, which was used for binding against Yst9.1 scFv, depicted in Figure 7. Figure 7 is a sensorgram showing the binding of 10 (M Yst9.1-L3-9 binding to immobilized Yst9.1 scFv. The control surface (BSA) data has been subtracted from the active surface (Yst9.1) data in this sensorgram. The rate constants were calculated by separate fitting of the association (fit shown) and dissociation data to a 1:1 interaction model. Figure 8 is a sequence diagram showing amino acid substitations for optional camelization according to one embodiment of the invention. Figure 9 is a diagrammatic representation of vector, SJFI, used to create the vector into which the library may be cloned. Figure 10 is a sequence diagram depicting the library construction strategy for library A6v H -Ll. Figure 11 is a sequence diagram depicting the library construction strategy for library A6V H -Lla. Detailed Description of Preferred Embodiments In a prefeπed embodiment, the invention is directed to a population of genetic packages having a genetically determined outer surface protein including genetic packages which collectively display a plurality of different ligand-binding molecules in association with the outer surface protein, each package including a nucleic acid construct coding for a fusion protein which is at least a portion of the outer surface protein and a variant of at least one soluble parental ligand-binding molecule derived from or having the amino acid sequence identified in Figure 2 (or a sequence preferably at least 80% homologous to the framework and conserved regions thereof), which includes at least one and preferably all of amino acids 6, 23 82a, 94 and 108 shown in Figure 2 (or conservative substitations of those residues which, if they do not improve, at least do not adversely affect the solubility properties of said parental V H ligand binding molecule), wherein at least part of the construct, preferably including at least part of the CDR3 identified in Figure 2, encodes or is biased in favor of encoding, the amino acid constitation of the parental ligand binding fragment such that the plurality of different ligand-binding domains are on the whole better adapted to be or better capable of being expressed as soluble proteins. The variant V H ligand-binding molecules are preferably characterized by a CDR3 having 16 to 33 amino acids. Preferably, the replicable genetic package is a recombinant phage and the heterogeneous population of replicable genetic packages collectively constitute a phage display library. In a further embodiment, the VL interface is engineered to avoid hydrophobic amino acids. In another embodiment, the VL interface is engineered for amino acids, which form weak interactions. In another embodiment the parental ligand binding molecule has a camelid type VL interface. In another embodiment, at least one of the VL interface amino acids are randomized or partially randomized in the construction of the library. Preferably the potential V H binding fragments include the entire FRl through to FR4 regions, although it is to be understood that partial deletions, for example, within CDR2, are contemplated to be within the scope of the invention. Preferably, CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the potential V H binding fragments. Preferably CDR3s of a variety of different lengths, from 18 to 28 amino acids, or from 20 to 25, or from 18 to 23, amino acids are predominantly represented in the library. In a preferred embodiment of the invention, the parental V H ligand-binding fragment is built on a human framework and preferably is the parental V H ligand-binding fragment identified in Figure 2 which has a CDR3 of 23 amino acids in length. More importantly, the invention encompasses a phage display library which is constructed using a parental V H ligand-binding molecule derived from native A6V H (Figure 1), or is built on any framework which is at least 80% (preferably 85%, more preferably 90 to 95%) homologous to the framework and other conserved regions of a fully human V H chain. The invention also contemplates that the parental V H binding-fragment, though not human, is adapted (egg. humanized) or adaptable (egg. to be adapted after selection of preferred binders) to a human framework. In another embodiment, the invention also contemplates the random, biased or fixed occurrence of features disclosed in the camelid literature, for example pairable cysteines in CDRl and CDR3 (optional) and/or the substitation of hydrophilic amino acids at least one of positions 44, 45, and 47 and preferably also positions 93 and 94 (Kabat numbering system). In a most prefeπed embodiment of the invention, the parental ligand-binding molecule is a N H fragment derived from a human IgM heavy chain, and preferably comprises FRl through FR4 of the N H chain. A partial sequence of the prefeπed antibody BT32/A6 (A6) is disclosed in U.S. Patent No. 5,639,863, incorporated herein by reference. The entire sequence is supplied now in Figure 1. In Figure 1, the CDR regions are demarcated. The amino acid residue numbers in Figure 1 and throughout the disclosure refer to the Kabat numbering system (Kabat et al. 1991, Sequences of Proteins of nmunological Interest, publication No. 91-3242, U.S. Public Health Services, NIH, Bethesda MD) except in the sequence listings and where explicitly stated or otherwise implied. Figure 1 coπesponds to SEQ. ID. NO. 1 (nucleic acid) and SEQ.ID. NO. 2 (amino acid). Figure 1 demarcates and labels regions CDRl (corresponding to SEQ. ID. NO. 3), CDR2 (SEQ. ID. NO. 4) and CDR3 (SEQ. ID. NO. 5). The A6 framework provides prefeπed solubility characteristics for creating dAb libraries. The term preferred solubility characteristics, as used herein, refers to at least one of the several, often correlated, characteristics including good yield, expression as a soluble product (as opposed to inclusion bodies) within the periplasm of the host organism, eg. Escherichia Coli, and a reduced tendency to dimerize and form other aggregates. The term "VH" is used to refer to the variable heavy chain domain and variants thereof made by routine skill in the art, including known forms, truncated and fusion protein forms thereof, which variants retain its essential binding characteristics. This term is used interchangeably with the terms such as V H binding fragment, V ligand binding fragment, dAb and immunoglobulin V H fragment, to which the same meaning is ascribed. The term "subsequence" is used to refer to a subset of nucleotides within a referenced nucleic acid sequence and includes a single nucleotide and consecutive and non-consecutive nucleotides within a sequence of greater than two consecutive nucleotides. The terms "polypeptide", "peptide" and "protein", unless the context implies otherwise, are used interchangeably herein, to refer to polymers of amino acid residues of any length. The term "combinatorial library" is used herein to refer to a set of molecules, typically belonging to a defined (narrowly or broadly) class comprising a substantial number of potentially useful variants, wherein the variations in the molecule represent a complete or partial set of permutations or combinations of at least some constituent elements of a reference molecule, which is typically a template or "parental" molecule, or simply the class itself. For clarity, in the case of polypeptides and nucleic acids, the constituent elements are amino acids and nucleic acid bases, respectively. As used herein, the phrase "in substantial part" refers to variations relative to a referenced molecule which do not significantly impair the "functionality" of that molecule. In the case of the parental ligand-binding molecule and variants thereof, functionality refers primarily to the solubility and binding characteristics of the molecule. Such variations (i.e. the referenced molecule in substantial part) can be tested systematically to assess their impact. In the case of framework regions, in contrast to CDR regions, due to the substantial conservation of the framework amino acid residues, a substantial part of the framework would preferably refer to at least 80% identity of the amino acid residues and more preferably an 85 to 100% identity, and even more preferably at least a 90% identity of the amino acid residues. However, it is understood that each of the previous percentages could be relaxed to discount instances where the absence of identity in a given residue, is due to a well recognized conservative amino acid substitation, or where a particular class of functionality is noted, e.g. hydrophilic, if the substitation is with a residue of the same class, i the case of CDR residues, these numbers could be considerably even more relaxed, and includes 50 to 100% homology, and all incremental percentages and all ranges of percentages therebetween. The term "in substantial part", in reference to portions of framework and CDR regions, also contemplates the possibility of additions and deletions in those regions which preferably do not impact the solubility and binding characteristics of the ligand-binding molecule in question. The term ligand-binding fragment is used broadly to define the whole or any part of an antibody that is capable of specifically binding to any ligand, in the broadest sense of the term ligand. An A6-based human heavy domain ligand-binding-fragment is well suited for the development of a combinatorial library (optionally a phage display library) that is used to generate soluble binding fragments that are useful for human diagnosis and therapy (due to limited HAMA response). These phage display libraries are used to selectively generate molecular probes that specifically interact with a ligand, including without limitation, natural and synthetic molecules and macromolecules and can be used in vitro (i.e., a diagnostic) and in vivo (i.e., a diagnostic and/or therapeutic) as indicators, inhibitors and immunological agents. The types of natural and synthetic molecules and macromolecules include but are not limited to: antibodies and fragments thereof; enzymes; cell receptors; proteins, polypeptides, peptides; polynucleotides, oligonucleotides; carbohydrates such as polysaccharides, oligosaccharides, saccharides; lipids; organic-based and inorganic-based molecules such as antibiotics, steroids, hormones, pesticides, herbicides, dyes, polymers. Conventional antibodies such as those found in human or murine species are composed of two identical light chains and two identical heavy chains. The combining sites of these antibodies are formed by association of the variable domains of both chains. This association is mediated through hydrophobic interactions at the interface. Structural and biochemical studies have shown that the heavy chain variable domain (V H ) provides most of the antigen-contacting residues (Padlan, 1994) (Chothia & Lesk, 1987) (Chothia, Novotny, et al, 1985). This finding has formed the basis for the development of single heavy domain antibodies (dAbs) - recombinant antigen binding fragments consisting of only the V H (Ward, Gussow, et al., 1989) (Cai & Garen, 1996). However, in the absence of their VL partners, V H S have been found to be insoluble, presumably because of the exposed hydrophobic VL interface (Ward, Gussow, et al., 1989). Heavy chain antibodies, found in camelids (Hamers, Atarhouch, et al., 1993) (Sheriff & Constantine,), lack light chains and as a result have variable domains that reflect the absence of a VL partner. Single domain antibodies derived from these antibodies are highly soluble and the structural basis of solubility has been partially elucidated. First, conserved human/murine interface residues such as Val37, Gly44, Leu45 and Trp47 are generally replaced in heavy chain antibodies by tyrosine or phenylalanine, glutamate, arginine or cysteine, and glycine, respectively. These mutations increase the hydrophilicity of the VL interface either by non-polar to polar substitations or, in a more subtle way, by inducing local conformational changes (Desmyter, Transue, et al., 1996) (Spinelli, Frenken, et al.,). This explanation is supported by experiments in which an insoluble human V H was made soluble by introducing the aforementioned mutations at positions 44, 45 and 47 (Davies & Riechmann, 1994). Second, in the solved structures of two camel dAbs, the CDR3s fold back on the V H surface, masking a significant surface area of the VL interface (Desmyter, Transue, et al., 1996)(Decanniere, Desmyter, et al., 1999). Several other features of V H Hs are noteworthy. One is the frequent occuπence of the cysteine residues in CDRl and CDR3 (Muylde mans, Atarhouch, et al., 1994) (Lauwereys, Arbabi, et al., 1998 (Vu, Ghahroudi, et al., 1997). While the location of the CDRl cysteine is typically fixed at position 33, that of the CDR3 cysteine varies. These two residues form a disulfide linkage between CDRl and CDR3 (Desmyter, Transue, et al., 1996) (Davies & Riechmann, 1996). In the crystal structure of a dAb-lysozyme complex, the disulfide linkage imparts rigidity on the CDR3 loop which extends out of the combining site and penetrates deep into the active site of lysozyme (Desmyter, Transue, et al., 1996). A second feature is the longer average length of the NπH CDR3, relative to human or murine V H S (Muyldermans, Atarhouch, et al., 1994). A longer CDR3, which is a feature of A6, increases the antigen binding surface and, to some extent, compensates for the absence of the antigen binding surface provided by the VL in conventional antibodies (Desmyter, Transue, et al., 1996). A third feature is the absence of the CDR3 salt linkage that is typically present in conventional antibodies and formed by arginine or lysine residues at position 94 and aspartate at position 101 (Desmyter, Transue, et al., 1996) (Muyldermans, Atarhouch, et al., 1994) (Spinelli, Frenken, et al., 1996) (Davies & Riechmann, 1996) (Chothia & Lesk, 1987) (Morea, Tramontano, et al., 1998). As antigen binding fragments, dAbs are an attractive alternative to scFvs because of their much smaller size and the fact that they demonstrate affinities comparable to those demonstrated by scFvs (Ward, Gussow, et al., 1989) (Spinelli, Frenken, et al., 1996) (Lauwereys, Arbabi, et al., 1998) (Davies & Riechmann, 1995) (Arbabi, Desmyter, et al., 1997) (Reiter, Schuck, et al., 1999). Smaller size is an advantage in applications requiring tissue penetration and rapid blood clearance. Smaller molecules also offer a tremendous advantage in terms of structural stadies (Davies & Riechmann, 1994) (Constantine, Goldfarb, et al., 1992 (Constantine, Goldfarb, et al., 1993). Phage antibody library construction is much simpler and more efficient if single domain antibodies (dabs) are used instead of Fabs or single chain Fvs. Randomization can be introduced at a much higher percentage of CDR positions without exceeding practical library size. The problem of shuffling original VL-V H pairings is also avoided. Camelid phage dAb libraries constructed from the V H H repertoire of camels immunized with target antigens have performed well (Arbabi, Desmyter, et al., 1997) (Lauwereys, Arbabi, et al., 1998) (Decanniere, Desmyter, et al., 1999). However, construction of libraries from immunized camels presents obvious problems. In addition, the non-human nature of products from these libraries limits their usefulness. Synthetic dAb libraries (Davies & Riechmann, 1995) (Reiter, Schuck, et al., 1999), particularly those based on a human NH framework, alleviate these problems. As described in detail in Example 1, a dAb phage display library, according to the invention was constructed by randomizing positions lOOi - lOOn of the parental N H ligand binding fragment identified in Figure 2 (A6N H -L1) The actual library size was 2.4 x 108 with 6.4 x 107 possible sequences. The sequences of 27 clones randomly picked from the unpanned library confirmed the integrity of the library (Table 1). Table 1. Sequences of the randomized region (lOOi-n) of randomly picked clones from the A6N H -Lla library. 1. DQFTHS 15 CVRGAE 2. SSMYGΝ 16 SPSLAA 3. IKMQQΝ 17 . HASGRS 4. SVDARD 18 GYMCSL 5. NSRFGA 19 HΝKDLA 6. GLGSPK 20 LADLYM 7. IDAKWA 21 WRRAHE 8. VSRFGA 22 SDLFAR 9. HCLPDG 23 VSRFGA 10. RWR7VP 24 RYRHST 11. VSRFGA 25 ARLAGP 12. LECEGC 26 SYRPYL 13. RΝVGAL 27 WLGΝS 14. RRSDYL As shown in Figure 5, this potential V ligand binding fragment shown has a substantially improved percentage of monomer relative to A6 wild-type . The A6N H -L1 library was panned against the anti-FLAG IgG M-2, HI 1 scFv and Brucella scFv. M-2 Results Three rounds of panning were performed. Twenty one clones from rounds 2 and 3 were sequenced (Table 2). The motifrecognized byM-2 is XYKXXD. As we observed previously with the camelized A6N H library, the epitope was preferentially positioned at the C-terminus of the randomized region with the D residue of non-randomized FDI sequence forming part of the epitope. As shown in Table, the sequences of the M-2 binders identified by phage ELIS A reasonably reproduce the motif recognized by M-2. Table 2. Sequences of M-2 binders identified by phage ELIS A. Sequence # of clones (A) deleted constructs ENQLQAS SGYYEDDYRLFDIWGQGTQNTNSS 6 ENQLQASGGGLNQP GYYDSSGYYKDLDTRFDIWGQGTQNTNSS 1 ENQLQASGGGLVQPGGSLRLS RLKNEYYDSSYYGDHYKWFDIWGQ GTQNTNSS 1 (B) Full length constructs (CDR3 only) DRLKNEYYDSSGYYRΝEYKEFDI DRLKNEYYDSSGYYNDEYKSFDI DRLKNEYYDSSGYYAGRYKDFDI DRLKNEYYDSSGYYRSDYKRFDI DRLKVEYYDSSGYYASHYKDFDI DRLKVEYYDSSGYYVDGYKDFDI DRLKVEYYDSSGYYTADYKMFDI DRLKVEYYDSSGYYDMDYKTFDI DRLKVEYYDSSGYYKS7YKSFDI DRLKVEYYDSSGYYDYKSQDFDI DRLKVEYYDSSGYYKΝWDSTFDI DRLKVEYYDSSGYYKDWDSSFDI DRLKVEYYDSSGYYKDGDSFFDI H11 Results Three rounds of panning were performed in each instance. No deleted sequences were observed for either antigen. Twenty -one HI 1 binders were sequenced. The sequence FSSP is present in 13 of the 21 Hll binders (Table 3). Table 3. CDR3 sequences HI 1 binders identified by phage ELISA. The randomized region is shown in bold. CDR3 # of clones (A) Hll scFv DRLKVEYYDSSG-YSFSSPFDI 7 DRLKVEYYDSSGYYYDFSSPFDI 3 DRLKVEYYDSSGYYNLFSSPFDI DRLKVEYYDSSGYYSEFSSPFDI DRLKVEYYDSSGYYSDFSSPFDI DRLKVEYYDSSGYYTDMSWEFDI DRLKVEYYDSSGYYDLGSWEFDI DRLKVEYYDSSGYYDYVSWEFDI DRLKVEYYDSSGYYDWGSWTFDI DRLKVEYYDSSGYYDG7TWDFDI DRLKVEYYDSSGYYWEGSGLFDI DRLKVEYYDSSGYYIWYSGLFDI DRLKVEYYDSSGYYSSWASAFDI Brucella scFv (Yst9.1) Clones obtained by panning against Yst9.1 scFv were selected for expression. Two clones had VSRFGA sequence. The VSRFGA dAb was designated Yst9.1-L3-9. The VSRFGA clone was expressed in good yield (approximately 18 mg/liter of bacterial culture) and was characterized by BIACORE. Yst9.1-L3-9 dAb Binding to Immobilized Yst9.1 scFv Following IMAC, the dAb was purified by Superdex 75 size exclusion chromatography prior to BIACORE analysis. It gave an elution profile that was similar to the parent A6V H -L1 molecule (Figure 6). For the determination of binding kinetics and affinities, the purified dAb was injected over active (1800 RU of Yst9.1) and reference surfaces (9400 RU of BSA) to generate the sensorgram and constants presented in Figure 7. The Yst9.1 surface was regenerated with 10 mM HC1 (6 sec contact). All proteins were immobilized on CM5 chips by amine coupling. A 2000 RU HI 1 scFv surface was also used as a reference. The dAb did not bind to BSA. The subtracted data, using the BSA surface as a reference, for dAb binding to Yst9.1 scFv fit quite well to a 1 : 1 interaction model. A KD of 117 nM was calculated. According to another embodiment of the invention, the parental ligand-binding fragment has additional amino acid substitations at VL interface which reduce the tendency to aggregation attributable to the "stickiness" of the V H dAb at this interface. For example, substitations at positions 44, 45 and 47 relative to the wild-type A6V H (Figure 1) are illustrated in Figure 8. According to another prefeπed embodiment of the invention, the parental ligand- binding fragment has a long CDR3. In a particularly preferred embodiment, the library is constructed using the A6V H -L1 parental V H ligand binding molecule as a template and preserves the entire length of this CDR3 and, additionally, at least one of positions 44, 45, 47 and 94 (and preferably all) ' is altered, (preferably including 44 or 45) to camelid type residues. Davies and Riechmann (1995) constructed a camelized dAb library by randomizing CDR3 amino acid residues but the library was ten times larger and yielded anti-hapten dAbs with dissociation constants in the range of 100-400 nM. However, the isolated anti-protein dAbs had weak affinity (Davies & Riechmann, 1995) (Davies & Riechmann, 1996). Therefore, a smaller library such as the one constructed here may therefore contain only weak anti-protein dAbs. The isolation of such dAbs would be difficult with monovalent display (Lowman, Bass, et al., 1991). In a phage vector format the dAb are displayed 3-5 copies and therefore there is potential for avidity which increases the likelihood of isolating weak binders (Nissim, Hoogenboom, et al., 1994). Additional embodiments of the randomization strategy for the libraries of the invention, are described below. The present inventors have also found a method of enhancing the probability that the binding fragments displayed in the library have characteristics which approximate the desired solubility characteristics found in the wild type binding fragment. During construction of the library, nucleotides of the variable region are added in a step-wise addition and by selecting a nucleotide ratio which is biased in favor of producing amino acids which reflect the DNA of the parental or wild type species. Thus, a method for biasing a library in favor of obtaining selected percentages of wild type amino acid residues is achieved by creating residue substitutions by using different spiking levels of the various dNTPs as described below. When creating a phage library, the randomization of amino acids is often achieved by DNA synthesis. A primer is annealed next to DNA encoding for the variable region, and nucleotides are randomly added to synthesize randomized variable regions. Normally, at the step of synthesizing the DNA used to produce the variable region of the phage library, one uses a nucleotide ratio of 1 : 1 : 1 : 1 , which generates a totally random variable region. By the present method, during synthesis of the variable region, the likelihood of achieving affinity or other desirable traits found in the wild type as follows. At each step of adding a nucleotide to the DNA variable region, one selects a dNTP ratio which is biased in favor of producing amino acids which reflect the DNA of the parental (wild type) species. Table 4 charts particular amino acid residues or sequences of residues and prefeπed types of amino acid substitations according to various examples of the invention to be defined hereafter. The selection of amino acids for randomization or partial randomization is based on adopting one or more of a variety of approaches including one of more of the following: 1. universal recognition of wild-type amino acids through a broad-based biasing in favour of the wild-type amino acids in one or more regions of interest (approximately 10%-90% biasing) in order to maintain the characteristics of the parental V H ligand- binding molecule; 2. selective recognition of amino acids that are important to maintain as wild-type through biasing (approximately 10-100%) in order maintain conserved or strategic regions of amino acid residues of the parental V H ligand-binding molecule; and 3. recognition of selected amino acids as important for intermolecular interaction and biasing those amino acids to wild-type and amino acids of the same type. Table 4 Unless otherwise necessarily implied as a result of logistical considerations, it is to be understood that the various embodiments which relate to choice of amino acids for random, biased or fixed substitation (specified in column 1) as well as the various embodiments related to types of substitations (column 2) are not mutaally exclusive. Moreover the various permutations and combinations of such substitations are hereby contemplated as embodiments of the invention. For example, substitations refeπed to in row a. (any one or more amino acids and preferably all amino acids of residues 100a- lOOh) #3 (at least approximately 50% wild-type amino acids) may combined with row b. (any one or more and preferably all of amino acids residues 100a, 100b, lOOg and lOOh) #2 (for instance, at least approximately 90% wild-type amino acids) so that, for instance, any 3 of the amino acids in 100a - lOOh are biased in favor of wild-type in approximately 50% of the variant V H ligand- binding fragments and 100a and 100b are biased in favor of wild-type in 90% of potential binding fragments. By necessary implication the three amino acids that are biased in favor of wild-type are not residues 100a and 100b, but they may be any other three residues. Accordingly, the broadest possible interpretation is to be given to the disclosure of the various combinations and permutations of the embodiments disclosed herein. Furthermore, it is to be understood that each of the various embodiments described herein are disclosed, except insofar as logistically impossible, in reference to each of the various aspects and definitions of the invention. Moreover, it is to be understood that phrases such as at least approximately 10%, or approximately 10-100% are intended to specify a preference for each of the unit percentages between about 7 and 100% that are practically achievable by oligonucleotide primer design and PCR amplification described herein below, as well as other well known PCR techniques and techniques of Controlled Mutation described in the art, and routine variations of such techniques. By the same token, phrases such as at least 80% are intended to specify a preference for each of the unit percentages between 80% and 100%. It is to be understood that biasing of a percentage less than 100% implies unless otherwise implied or stated that the remaining percentage is tally randomized. Furthermore, it is to be understood, for example, that 90% biasing in favor of wild-type amino acids at a given amino acid position is to be approximated by controlling the percentage amounts of each of the three relevant nucleotides (so that, for example, the product of the probabilities of occuπence of the three desired nucleotides in sequence in the growing chain is 90%) so as to supply 90% of coπect coding triplet(s) and a total of 10% of random coding triplets, having regard to the degeneracy of the genetic code (for example if two different coding triplets result in a given amino acid, then the sum of the probabilities of achieving those two triplets will have to equal 90%). This is preferably accomplished on an amino acid by amino acid basis so that, for example the probability of achieving two and three wild-type amino acids in sequence, in the case of 90% biasing is 0.81 and 0.73, respectively, etc. It is to be understood that this high level of biasing may be suitable only for part of the coding sequence into which variability is introduced and that higher levels of biasing are acceptable, when for example substantially all of the amino acids of a long CDR3 are biased, as disclosed in one of embodiments herein. Accordingly there is a balance to be struck between a large diverse library and biasing for multifactorial characteristics such as solubility. Nevertheless it is contemplated that the library produced may be a pooled library in which several libraries each having varying degrees of biasing to wild-type, for example, 60%, 50%, 40% and 30%, are pooled together to obtain the both desired variability and similarity. The prefeπed parental binding-fragment may be engineered to maximize the desired characteristic (e.g. solubility, intermolecular interaction) and then made the subject of libraries with varying degrees of biasing. In this connection, the library could be biased to be rich in amino acids, which are highly soluble. It is to be understood that both arms (halves) of the prefeπed longer loop forming CDR3s may be biased to amino acids that are favored for intermolecular interaction, preferably charged amino acids, so as to provide a method of generating, in addition to loop size, varying loop structures. This bias may be systematically introduced or systematically reduced by randomization, in cooperating pooled libraries having varying degrees of biasing. With respect to the application of these methods to parental VH, preferably, CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the variant VH ligand-binding fragments. Preferably CDR3s of a variety of different lengths, from 18 to 25 amino acids, or, from 18 to 23 amino acids are predominantly represented in the library. Although the term "predominant" ordinarily implies a majority representation of the specified long CDR3 variant VH ligand-binding fragments, the invention also contemplates an even less substantial representation, especially within a reasonably large size library (>10 7 ). Preferably, the specified long CDR3 variant VH ligand-binding fragments have a majority representation within the library and more preferably an even greater or exclusive representation. Optionally, the parental V H ligand-bindingmolecule is reduced in size and the parental VH ligand-binding molecule is optionally modified by deleting a portion of the CDR2. In another embodiment, CDR3s of the same length as that of the parental VH ligand-binding molecule are predominantly or exclusively represented in the variant V H ligand-binding fragments. In another aspect, the CDR3 region is specifically retained along with human sequence elements of other regions that confer favorable characteristics solubility, to create a phage display library having favorable characteristics of solubility, preferably when compared with variant V H ligand-binding fragments that have fully randomized hypervariable regions (particularly CDR3). In particular, the present inventors have found that favorable solubility characteristics of a parental V H ligand-binding molecule can be maintained in the population of variant V H ligand-binding fragments in the course of randomizing the hypervariable regions by biasing all or selected amino acids residues to wild-type and/or biasing in favor of amino acids residues that favor certain or a variety of types of intermolecular interaction. This is respectively accomplished by increasing the percentage amounts of nucleotide bases that represent wild-type amino acids and/or amino acids that provide favorable intermolecular interactions, during the randomization procedure e.g. site directed PCR mutagenesis. Thus, variant N ligand-binding fragments having relatively long CDR3s of varying lengths are produced by randomly or partially randomly inserting varying numbers of nucleotide triplets in any part of a randomized portion of the parental VH framework. Primers of the desired length and nucleotide composition are synthesized followed by PCR amplification. Desired randomization can be achieved by biasing nucleotide composition of the primer. The production of displays of long CDR3 variant binders may also be accomplished by pooling several libraries of variant V H ligand-binding fragments having randomized or partially randomized CDR3s of different respective uniform lengths. These strategies are not mutaally exclusive. The additional following terms are used herein as follows, unless the context logically implies otherwise: "Biasing", "biased in favor of and related forms of these terms are generally intended to refer to weighting in the course of introducing variation in the parental ligand-binding molecule. "Homologous" or "homology" as used herein refers to "identity" or "similarity" as used in the art, meaning relationships between two or more polynucleotide or amino acid sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Both identity and similarity can be readily calculated (Lesk, A. M., ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, D. W., ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, 1993; Griffin, A. M., and Griffin, H. G., eds., Computer Analysis of Sequence Data, Part I, Humana Press, New Jersey, 1994; von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, 1987; and Gribskov, M. and Devereux, J., eds., Sequence Analysis Primer, M Stockton Press, New York, 1991). While there exist a number of methods to measure identity and similarity between two polynucleotide sequences, both terms are well known to skilled artisans (von Heinje, G., 1987;Gribskov, M. and Devereux, J., 1991; and Carillo, H., and Lipman, D., 1988). Methods commonly employed to determine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D. (1988, SIAMJ. Applied Math., 48: 1073). Methods to determine identity and similarity are codified in computer programs. Prefeπed computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al.(1984), Nucleic Acids Research 12(1): 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al.(1990), J. Molec. Biol. 215: 403). "Percent homology" or "% homologous" or related terms include both of the following interpretations / methods of calculation: 1) an approximate percentage of the sequence referenced in terms of the number of common residues (e.g. 80% of 11 is understood to be an approximation insofar as application of the percentage does not yield a unit number of residues, in which case both the immediately higher number and immediately lower unit numbers, 9 and 8 respectively, are deemed to be covered); 2) the percentage of binding fragments theoretically achievable that have the full wild-type sequence, which is calculated as a product of the probabilities that the wild-type amino acid will occur at a given amino acid position. "Conserved" regions refer to those which are commonly found in at least other antibodies of the same type or in at least the same species of mammal. "Wild-type" refers to the parental binding-fragment, which may be a variant of the natural or to the native A6 N H parental ligand-binding fragment, depending on the context. "Step-wise" refers to the addition of, for example, nucleic acids, in a manner such that the quantity of nucleic acids added at each step is rigorously control, usually one nucleic acid at a time. "Spanning" does not preclude deletions or additions within the parental N H binding- fragment that are not inimical to the operation of the invention. "Camelid type" refers specifically to one or more features of the camelid NL interface. "Soluble" includes the generally ascribed meaning in the art and without limitation includes (based on solubility coπelated phenomena) the relative amounts of naturally-folded recombinant protein released from the cell. "Percent biasing" or "% of binding fragments" (or "biasing 10-100%", etc.) refers to biasing on an individual amino acid basis (though other techniques to accomplish the same effect might apparent to those skilled in the art). Similarly, the specification that wild-type amino acids occur at a specified position or series of positions in, for example, at least approximately 50% of potential binding fragments is intended to mean both that 50% biasing is sought at a given such position or that a total of 50% of the coπect nucleotide triplets are represented. "Approximately" in reference to percentages is intended to accommodate attrition of various desired variant N H ligand-binding fragments, the assumption that the probabilistic outcomes will not be achieved in practice and that certain variation in methods to accomplish the specified results is deemed to be suitable. The term 50% in reference to an uneven number of amino acids residues means that either one more or one less than half of the amino acids is refeπed to. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D.M. Wei & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Cuπent Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991). These references are incorporated herein by reference. These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. Recombinant genetic techniques have allowed cloning and expression of antibodies, functional fragments thereof and the antigens recognized. These engineered antibodies provide novel methods of production and treatment modalities. For instance, functional immunoglobulin fragments have been expressed in bacteria and transgenic tobacco seeds and plants. Skeπa (1993) Curr.Opin. Immunol. 5:256:262; Fiedler and Conrad (1995) Bio/Technology 13:1090-1093; Zhang et al. (1993) Cancer Res. 55:3384-3591; Ma et al. (1995) Science 268:916; and, for a review of synthetic antibodies, see Barbas (1995) Nature Med. 1 :836-839. These and more current references describing these techniques, which these references, particularly those well known to persons practicing in the relevant arts, are hereby incorporated herein by reference. Nucleotide sequences can be isolated, amplified, and processed by standard recombinant techniques. Standard techniques in the art include digestion with restriction nucleases, and amplification by polymerase chain reaction (PCR), or a suitable combination thereof. PCR technology is described in U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston (1994). In addition to the specific PCR methods of biasing to wild-type A6 amino acid residues detailed below, it is possible to produce multiple different oligonucleotide primers consisting of specified amino acid residues (one or more) of the wild-type A6 molecule (e.g. CDR3 residues), mixing these in appropriate concentrations with a completely randomized (e.g. CDR3) oligonucleotide primer and subjecting the mixture of oligonucleotide primers to PCR. This will result in a biased phage library population of one's choosing (i.e. the amounts of the selectively randomized and totally randomized primers in the mixture will determine the per cent of each CDR3 representation in the library). Polynucleotides comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be introduced into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell by standard methods. See, e.g., Sambrook et al. (1989). RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerase. Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and are not described in detail herein. See e.g. Gacesa and Ramji, Vectors, John Wiley & Sons (1994). Phage display techniques are generally described or referenced in some of the preceding general references, as well as in U.S. Patent Nos. 4,593,002; 5,403,484; 5,837,500; 5,571,698; 5,750,373; 5,821,047; 5,223,409 and 5,702,892. "Phage Display of Peptides and Proteins", (Kay, Brian K. et al., 1996); "Methods in Enzymology", Vol. 267 (Abelson, John N., 1996); "Immunology Methods Manual", (Lefkovits, Ivan, 1997); "Antibody phage display technology and its applications", (Hoogenboom, Hennie R. et al., 1998). Immunotechnology 4 p .1-20; Cesareni G et al. Phage displayed peptide libraries. Comb Chem High Throughput Screen. 1999 Feb;2(l):l-17; Yip, YL et al. Epitope discovery using monoclonal antibodies and phage peptide libraries. Comb Chem High Throughput Screen. 1999 Jun;2(3): 125-38; Rodi DJ et al. Phage-display technology—finding a needle in a vast molecular haystack. Curr Opin Biotechnol. 1999 Feb; 10(1): 87-93. Generally, DNA encoding millions of variants of a parental binding-fragment can be batch-cloned into the phage genome as a fusion to the gene encoding one of the phage coat proteins (pITI, pVI or pVIH). Upon expression, the coat protein fusion will be incorporated into new phage particles that are assembled in the bacterium. Expression of the fusion product and its subsequent incorporation into the mature phage coat results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle. This connection between ligand genotype and phenotype allows the enrichment of specific phage, e.g. using selection on immobilized target. Phage that display a relevant ligand will be retained, while non-adherent phage will be washed away. Bound phage can be recovered from the surface, reinfected into bacteria and re-grown for further enrichment, and eventually for analysis of binding. The success of ligand phage display hinges on the combination of this display and enrichment method, with the synthesis of large combinatorial repertoires on phage. While the use of phage is described as an embodiment for the production of libraries for displaying, and selecting particular binding fragments, it is to be understood that and suitable genetic package may be used for the production of libraries of the invention. Such suitable genetic packages include cells, spores and viruses (see US Patent No. 5,571,698), or any other suitable replicable genetic packages. With respect to cell based approaches, another popular method of presenting a library is the two-hybrid system (Feilds and Sternglanz, 1994, Trends in Genetics 10:286-292). Those skilled in the art will appreciate that in vitro systems (non-cell based) may be equally applicable to the methods of the present invention, for example ribosome display (Hanes et al., 1998) or RNA-peptide fusion (Mattheakis et al., 1994, Proc Natl Acad Sci USA 91 :9022-26; Hanes et al., 1999, Curr Top Microbiol Immunol 243:107-22). Ribosome display is a well documented technique that may be useful for generating 1 libraries. This entirely in vitro method allows for libraries with a diversity of >10 . In this method, a peptide is displayed on the surface of a ribosome that is translating it. Briefly, a library of mRNA molecules (we could start with A6) is translated in vitro translation system to the 3' end, such that the ribosome does not fall off. The protein emerges from the ribosome in such a way that it can fold, but does not fall off. In some instances, there is an additional folding step in an oxiding environment (important for proteins with disulfide bonds). The whole complex of folded protein, ribosome and mRNA, which is stable for several days, can then be panned against a ligand that is recognized by the translated protein. (For example, the translated protein could be an antibody and the ligand is its antigen). The mRNA can then be amplified by reverse transcription and PCR. This technique has been used to successfully generate scFv antibody fragments with high affinity for their target. Reference is made to Hanes,J., Jermutus,L., Weber-Bornhauser,S., Bosshard,H.R. & Pluckthun,A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA 95, 14130-14135 (1998); SchaffitzeLC, Hanes ., Jermutus,L. & Pluckthun,A. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. Journal of Immunological Methods 231, 119-135 (1999); He,M. et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. Journal of Immunological Methods 231, 105-117 (1999); Roberts,R.W. Totally in vitro protein selection using mRNA-protein fusions and ribosome display. Current Opinion in Chemical Biology 3, 268-273 (1999); Williams,C. Biotechnology match making: screening orphan ligands and receptors. Current Opinion in Biotechnology 11, 42-46 (2000); Mattheakis,L.C, Bhatt,R.R. & Dower,WJ. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022-9026 (1994). Construction of A6NH-L1 Library Example 1 A dAb phage display library was constructed employing the Vnportion of A6 as a starting template and amino acid substitations at positions 6, 23, 82a, 93 and 108, as shown in Table 5 below. In addition, silent mutations were introduced in codons for amino acids 3-16 (FRl) to remove a putative recombination site. All mutations were introduced by splice overlap extension - PCR (SOE). The modified V H was used as a scaffold for complete randomization at positions 1 OOi- 1 OOn located at the carboxy terminal of the CDR3 of A6. Table 5. A6V H -L1 Template Position and Amino Acid Identity Position 6 23 82a 93 108 Wild Type A6V H E S S V T Modified A6VH A A Ν A Q Removal of Putative Recombination Site The codons for amino acids 3 - 16, which surround the recombination site, were changed. Using the Chi.R/FP and Chi.F/RP primer pairs (Table 6) and pSJF-A6V H plasmid as template, 5' and 3' fragments were synthesized by PCR in a total volume of 50 μl containing 10 pmol of each primer, 2mM of each of the four dΝTPs, IX buffer and 2.5 units of AmpliTaq™ DΝA polymerase (Perkin Elmer). The PCR protocol consisted of an initial denaturation step at 94°C for 3 min followed by 30 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and a final extension step at 72°C for 10 min. The two fragments were gel purified using the QlAquick Gel Extraction™ kit (Q1AGEΝ) and a larger construct was assembled from the 5' and 3' fragments by performing splice overlap extension (SOE) PCR using RP and FP. Briefly, the reaction vial containing both 5' and 3' fragments, 200 μM each of the four dΝTPs, 5 μl 10X buffer (ΝEB), and 2 units of Vent DΝA polymerase (NEB) were subjected to 7 cycles of 1 min at 94°C and 2.5 min at 72°C. To amplify the assembled construct, RP and FP primers were added at a final concentration of 1 pmol/μl and the mixture was subjected to 30 cycles of 1 min at 94°C, 30 sec at 55°C, and 1 min at 72°C. The amplified product was purified (Ql Aquick PCR Purification™ kit) and used as template for the mutagenesis steps. Sequencing revealed that the putative recombination sequences had been removed. Modification of Positions 6, 23, 82a, 93 and 108 Employing SOE, positions 23, 82a, 93 were mutated initially (Table 5). Using the Chi template, three fragments were synthesised by PCR using the A6V H .S23A.R/A6V H .S82aN/V93A.F (fragment 1), A6V H .S23A.F/RP (fragment 2) and A6V H V93A.R/FP (fragment 3) primer pairs (Table 6). The first two fragments were assembled by SOE using RP/A6V H -S82aN/V93A and the resultant fragment was spliced to the third fragment using RP/FP primers. Sequencing revealed the desired mutations at positions 23, 82a and 93. This product in tarn was used as template for the final round of SOE experiments to alter positions 6 and 108. First, two fragments were synthesized using the A6V H .E6A.R/A6V H .T108Q.F and A6V H .E6A.F/RP primer pairs followed by SOE using RP/A6V H .T108Q.F primers. The final construct was purified, digested with BamRI and EcoRl, purified again and ligated to Rα HI/EcoRI-restricted expression plasmid. The colonies were then screened to identify clones containing the mutated A6V H . TABLE 6. PRIMERS USED CHI SITE REMOVAL AND MUTAGENSIS OF A6V H . Chi.R CAATTACAAGAAAGTGGTGGCGGACTGGTGCAACCAGGAGGTTCCCTGAGACTC CW.F ACTTTCTTGTAATTGGACCTCGGCCTGCGC A6VH.S23A.R CTCTCCTGTGCTGCCTCTGGA A6VH.S23A.F TCCAGAGGCAGCACAGGAGAG A6VH.S82aN/V93A.F CGCACAGTAATACACAGCCGTGTCCTCAGCTCTCAGACTGTTCATTTGAAGATA A6VH.V93A.R GTGTATTACTGTGCGAAAGACAGG A6VH.E6A.R CAATTACAAGCTAGTGGTGGC A6VH.E6A.F GCCACCACTAGCTTGTAATTG A6VH.T108Q.F TATGGATCCTGAGGAGACGGTGACCTGTGTCCCTTGGCC A6VH.ApalI.R CATGACCACAGTGCACAGGAGGTCCAATTACAAGCTAGA A6VH.NOT.T108Q CGATTCTGCGGCCGCTGAGGAGACGGTGACCTGTGTCCCTTGGCCCCAGATATC RP GCGGATAACAATTTCACACAGGAA FP CGCCAGGGTTTTCCCAGTCACGAC Cloning, Expression and Evaluation of Dimer/Multimer Formation The modified A6VH, designated A6VH-L1 was cloned into a vector for expression in E. coli using EcoRl and BamHl. Thirty ml of LB containing 100 ug ml ampicillin was inoculated with a single colony harboring pSJF2-dAb and the culture was shaken at 240 rpm at 37°C overnight, hi the morning the entire overnight culture was used to inoculate 1 liter of M9 medium supplemented with 5 μg/ml vitamin Bl, 0.4% casamino acids and 100 μg/ml ampicillin. The culture was shaken at room temperature for 30 hr at 160 rpm and subsequently supplemented with 100 ml of lOx induction medium and 100 ul of 1M isopropylthio-/3-D-galactoside. The culture was shaken for another 60 hr, the periplasmic fraction was extracted by osmotic shock method (Anand et al., 1991), and the presence of dAb in the extract was detected by Western blotting (MacKenzie 1994). The periplasmic fraction was dialyzed extensively in 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N' [2- ethanesulfonic acid]) buffer pH 7.0, 500 mM NaCl. The presence of the dAb C-terminal His 5 tag allowed a one step protein purification by immobilized metal affinity chromatography using HiTrap Chelating™ column (Phamacia). The 5-ml column was charged with Ni 2+ by applying 30 ml of a 5 mg/ml NiCl 2 .6H 2 O solution and subsequently washed with 15 ml deionized water. Purification was carried out as described (MacKenzie, 1994) except that the starting buffer was 10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the bound protein was eluted with a 10-500 mM imidazole gradient. The purityof the protein was determined by SDS-PAGE (Laemmli). To detect the presence of dimer/multimer dAb in the protein preparation, gel filtration chromatography was performed using Superdex75 (Pharmacia) as described (Deng et al., 1995). Library Construction A PCR product was generated using A6V H -L1 as template and primers RP and A6V H .Rndml00i-n.F, CCTTGGCCCCAGATATCAAA6[(A/C)NN]GTAATAACCACTACTATC. The latter primer was degenerate in the region encoding residues lOOi-n. A second PCR employed 180 pmol of the above product and 100 pmol of each of the two primers AόVπ-ApaLR, CATGACCACAGTGCACAGGAGGTCCAATTACAAGCTAG, and A6V H .NotTl 08Q.F, CGATTCTGCGGCCGCTGAGGAGACGGTGACCTGTGTCCCTTGGCCCCAGATATC. The second set of primers are complimentary to the 5' and 3' ends of the dAb genes and incorporate ApaR and Notl restriction sites (underlined sequences) at the end of the amplified genes. The amplified products were purified, cut sequentially with ApaR and Notl restriction endonucleases, purified again, and ligated to the vtøα/I/Notl-treated fd-tet phage vector. Following this, 1.5 μg of the desalted ligated product was mixed with 40 μl of competent E. coli strain TGI and the cells were transformed by electroporation. Transformation, library phage amplification and purification and library size determination were performed as described below. The randomization strategy is depicted in Figure 11. Library size determination. To determine the size of the library, immediately following the transformation and after the addition of the SOC medium an small aliquot of the electroporated cells were serially diluted in exponentially growing TGI cells. Two hundred μl of the diluted cells was mixed with 3 ml of 50°C agarose top and immediately poured onto 2xYT plates pre- warmed to 37°C. Plates were incubated overnight at 37°C and the number of plaques were used to determine the size of the library. Panning Panning was performed using the Nunc-hnmuno MaxiSorp™ 8-well strips (Nunc). Briefly, the wells were coated overmght by adding 150 μl of 100 μg/ml antigen in PBS. In the morning, they were rinsed three times with PBS and subsequently blocked with 400 μl PBS-2% (w/v) skim milk (2% MPBS) at 37 ° C for 2 hr. The wells were rinsed as above and 10 transducing units phage in 2% MPBS were added. The mixture was incubated at room temperature for 1.5 hr after which the unbound phage in the supernatant was removed. The wells were rinsed 10 times With PBS-0.1 % (v/v) Tween 20 and then 10 times with PBS to remove the detergent. The bound phage was eluted by adding freshly prepared 200 μl 100 mM triethylamine, pipetting the content of the well up and down several times and incubating the mixture at room temperature for 10 min. The eluted phage was transfered to a tube containing 100 μl 1 M Tris-HCl, pH 7.4 and vortexed to neutralize triethylamine. Following this, 10 ml exponentially growing TGI culture was infected with 150 μl eluted phage by incubating the mixture at 37 ° C for 30 min. Serial dilutions of the infected cells were used to determine the titer of the eluted phage as described in the previous section. The remaining of the infected cells were spun down and then resuspend in 900 μl 2xYT. The cells were mixed in 300 μl aliquots with 3 ml agarose top and the phage propagated on the plates overmght at 37 ° C. In the morning the phage was purified, the titer was determined, and a total of 10 1 transducing units phage were used for further rounds of selection. Example 2 A library (A6Vπ-Lla) in which residues lOOi and lOOn were deleted and residues 95 -lOOh were randomized with 50% biasing in favour of the parental amino acid constitation, was also constructed. The procedure for A6Vκ-Lla construction was identical to that for A6V H -L1 except that the randomization primer used in the first PCR step was A6N H -95- lOOhRndm CCCTTGGCCCCAGATATCAAA14[(A/C)ΝΝ]TTTCGCACAGTAATACAC. The sequence of the A6NH for this library is shown in Figure 12. Example 3 A6N H -L2, a parental N H ligand binding molecule in which each of positions 6, 23, 74, 82a, 83, 84, 93 and 108 were modified as shown in Figure 3 relative to the native A6N H (Figure 1), was also constructed. Mutations at positions 74, 83 and 84 were introduced with a single mutagenic primer, A6N H .S74A.F, (5'CATTTGAAG- ATACAGAGTGTTCTTGGCATTGTCTCT3 '), which when used together with RP (see Example 1 above) generated an Ν-terminal fragment. A second primer, A6NH.R83K/A84P.R, (5 'TATCTTCAAATGAACAGTCTGAAAC- CAGAGGACACGGCT3'), together with FP (see Example 1 above) generated a C-terminal fragment. The two fragments were joined by SOE, using RP and FP as primers, to give the gene encoding A6N H -L2. This molecule showed slightly better levels of monomer production when expressed and assessed by size exclusion chromatography when compared with A6N H -Ll. Table 7. Amino acid identity at positions 6, 23, 74, 82a, 83, 84, 93 and 108 for A6V H and the mutants of A6V H .3 Amino acid Identity Kabat # 6 23 74 82a 83 84 93 Ϊ08 A6VH E S S S R A V T A6VH-L1 A A S Ν R A A Q A6VH-L2 A A A Ν K P A Q Example 4 - Surface Plasmon Resonance Binding stadies were performed using BIACORE Upgrade (Biacore Inc., Piscataway, NJ) as described (Jonsson et al., 1991). Approximately 14, 000 RU of anti-FLAG M2 IgG or control IgG were immobilized on CM5 sensor chips by amine coupling. Single-domain antibodies were passed over the sensor chips surfaces in 10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 (Biacore Inc.) at 25°C and at a flow rate of 5 μl/min. Sensorgram data were analyzed using the BIAevaluation 3.0 software package (Biacore Inc.). Example 5 - Enzyme-Linked Immunosorbent Assay (ELISA) Nunc-hnmuno MaxiSorp™ plates (Nunc) were coated overnight at 4°C with 150 μl of 10 μg/ml of 3B1 scFv or BSA in PBS. The contents were removed and the plates were tapped on a paper towel to remove any liquid remaining in the wells. The wells were blocked by adding 300 μl of 2% MPBS and incubating for 2 hr at 37°C. The contents of the wells were emptied as before, 100 μl of purified dAb phage in 2% MPBS was added, and the wells were incubated at room temperature for 1.5 hr. The contents were emptied again and the wells were washed 5 times with PBS-0.05% (v/v) Tween 20 and subsequently blotted on a paper towel to remove any remaining wash buffer. One Hundred μl of recommended dilution of HRP/Anti-M13 monoclonal antibody conjugate (Amersham Pharmacia Biotech) in 2% MPBS was added and the wells were incubated at room temperature for 1 hr. The wells were washed six times as before and the binding of dAb to the antigen was detected colorimetrically by adding 100 μl of equal mixtures of TMB Peroxidase Substrate and H 2 O (Kirkegaard and Perry Laboratories, Gaithersberg, MD, USA) at room temperature for several minutes. The reaction was stopped by adding 100 μl of 1 M H 3 PO 4 and the ^o was measured by DYNATECH MR5000 plate reader (Dynatech Laboratories, Chantilly, VA, USA). Example 6 - Introducing Genetic Variation into the Sequence Corresponding to the A6 Heavy Chain CDR3 Region - Randomized Residues Oligonucleotides comprising randomly mutated CDR3 regions were prepared on an Applied Biosystems 394 DNA synthesizer as described above. 1. Production of 23 randomized residues (CDR3 1-23): The anti-codon formula [(A C)NN] is used resulting in a reduction in possible codon usage from 64 to 32 and reduces the number of possible stop codons. Position one, therefore, comprises only A and C in the synthetic reaction mixture. For complete randomization of the second and third positions of the codons the dNTP mixture comprise 25% each of A,G,C and T. The 3' oligonucleotide randomizing primer was designed such that the last 15 nucleotides of framework 3 and the first 17 nucleotides of framework 4 were kept constant for hybridization. The nucleotides encoding the intervening amino acids, namely amino acids 1 -23 of the CDR3 region were randomized using the following primer: 5' (GTTGTCCCTTGGCCCCA n[(A/C)NN]TTTCACACAGTAATA] 3' (Where n=23,antisense strand). Using a 50% A and 50% C for the first nucleotide position for each anti-codon triplet and 25% each of A, C, G, and T for the second and third nucleotide positions for n=23, complete randomization of the 23 amino acids of the A6 CDR3 is achieved. 2. Synthesis of CDRs comprising 15-23 residues The primers are adapted by reducing n to 15-23 in the above primer formulae whilst keeping the flanking nucleotides constant. 3. For synthesis ofCDR3s comprisins 24-33 residues The primers would be adapted by increasing n to 24-33 in the above primer formula while keeping the flanking nucleotides constant. Example 7 - Selective Randomization Biasing for 50% Homology to Parental Tyrosine To achieve approximately 50% homology to wild type at any one position in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer, the following example would be used. In the case of tyrosine, which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the antisense strand. First anticodon nucleotide position: 80% of A and 20% of C is added to the dNTP solution, and G and T are not added to reduce codon degeneracy. Second anticodon nucleotide position: 80% T and approximately 6.67% of C, 6.67 of A and 6.67% of G. Third anticodon nucleotide position the mixture: 80% of A and approximately 6.67% of T and 6.67% of G and 6.7% C. The calculated probability of tyrosine would thus be 0.8x 0.8 x 0.8 x 100% = 51.2 %. Thus approximately 51% of the chains of the library will contain a wild-type A6 tyrosine in that specified position. Example 8 - Selective Randomization Biasing for 50% Homology to Parental Serine Using the same strategy in order to achieve approximately 50% homology to wild type serine at one or more positions, the following example is useful. Using only A and/or C in the first anticodon position the amino acid serine could have two codons these are AGT, TCT and TCG (antisense ACT, AGA and CGA, respectively). The nucleotide spiking levels would be as follows: First anticodon nucleotide position: 50% A and 50% C. Second anticodon nucleotide position: 35.35% C, 35.35% G, 14.65% A and 14.65% T Third anticodon nucleotide position: 35.35% A, 35.35% T, 14.65% C and 14.65% G. The probability of producing serine for any given fragment, using this strategy is (1 x [0.3535+0.3535] x [0.3535+0.3535] x 100% = 50 %. Thus, approximately 50% of the chains will have a serine in the selected position. Example 9 - Selective Randomization Biasing for 50% Homology to Parental Serine To achieve approximately 10% homology to wild type at any one position in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer, the following example can be used. In the case of tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the anti sense strand. First anticodon nucleotide position: 47% of A and 53% of C is added; G and T are not added to reduce codon degeneracy. Second anticodon nucleotide position: 47% T and approximately 17.67 % of C, 17.67 of A and 17.67% of G. Third anticodon nucleotide position: 47% of A and approximately 17.67% of T and 17.67% of G and 17.67% C. The calculated probability of tyrosine is thus 0.47x 0.47 x 0.47 x 100% = 10.4 %. Thus approximately 10% of the chains of the library will contain a wild-type A6 tyrosine in that specified position. Example 10 - Selective Randomization Biasing for 50% Homology to Parental Serine To achieve approximately 90% homology to wild-type amino acids at any positions in the A6 dAb CDR3 region during antisense synthesis using the DNA synthesizer , the following example would be used. In the case of tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows: First anticodon nucleotide position: 97% of A and 3% of C is added, G and T are not added to reduce codon degeneracy. For this reason, only A and C are used in the first anticodon position for all 20 naturally occurring amino acids. Second antcodon nucleotide position: 97% T and approximately 1 % of C, 1% of A and 1% of G. Third anticodon nucleotide position: 97% of A and approximately 1% of T and 1% of G and l% C. The calculated probability of tyrosine would be 0.97x 0.97 x 0.97 x 100% = 91.3 %. Thus approximately 90% of the chains of the library will contain a wild-type A6 tyrosine in that specified position. Using the approaches in the examples above, approximately 10 % to approximately 90 % of wild type amino acid representation at one or more specified amino acid residues in the A6 CDR3 can be calculated and applied to the DNA synthesizer. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Certain adaptations and modifications of the invention will be obvious to those skilled in the art. Therefore, the presently discussed embodiments are considered to be illustrative and not restrictive. It is understood that the claims may refer to aspects or embodiments of the invention that are only mferentially refeπed to in the disclosure. All references disclosed in this application are hereby incorporated by reference. Additional References: Anand, N. N., Dubuc, G., Phipps, J., MacKenzie, C. R., Sadowska, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1991). Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen. Gene 100:39-44. Arbabi, G. M., Desmyter, A., Wyns, L., Hamers, R., and Muyldermans, S. (1997). Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521-526. Bodenhausen, G. and Ruben, D. J., Chem. Phys. Lett., 69, 185-188, 1980. Cai, X. and Garen, A. (1996). A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient. Proc Natl Acad Sci USA 93:6280-6285. Chothia, C and Lesk, A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. JMol Biol 196:901-917. Chothia, C, Novotny, J., Bruccoleri, R., and Karplus, M. (1985). Domain association in immunoglobulin molecules. The packing of variable domains. JMol Biol 186:651-663. Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and Winter, G. (1991). Making antibody fragments using phage display libraries. Nature 352:624-628. Constantine, K. L., Goldfarb, V., Wittekind, M., Anthony, J., Ng, S. C, and Mueller, L. (1992). Sequential IH and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain. Biochemistry 31:5033-5043. Constantine, K. L., Goldfarb, V., Wittekind, M., Friedrichs, M. S., Anthony, J., Ng, S. C, and Mueller, L. (1993). Aliphatic IH and 13C resonance assignments for the 26-10 antibody VL domain derived from heteronuclear multidimensional NMR spectroscopy. JBiomolNMR 3:41-54. Dan, M., Earley, E. M., Griffin, M. C, Maiti, P. K., Prashar, A. K., Yuan, X. Y., Friesen, A. D., and Kaplan, H. A. 1995. Human monoclonal antibody BT32/A6 and a cell cycle- independent glioma-associated surface antigen. J. Neurosurg. 82, 475-480. Davies, J. and Riechmann, L. (1994). 'Camelising' human antibody fragments: NMR studies on VH domains. FEBS Lett 339:285-290. Davies, J. and Riechmann, L. (1995). Antibody VH domains as small recognition units. Biotechnology N Y 13:475-479. Davies, J. and Riechmann, L. (1996b). Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding. Immunotechnology 2:169- 179. Davies, J. and Riechmann, L. (1996a). Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. Protein Eng 9:531-537. Decanniere, K., Desmyter, A., Lauwereys, M., Ghahroudi, M. A., Muyldermans, S., and Wyns, L. (1999). A single-domain antibody fragment in complex with RNase A: non- canonical loop structures and nanomolar affinity using two CDR loops. Structure 7:361-370. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. and Bax, A., J. Biomol. NMR 6, 277-293, 1995. Deng, S. J., MacKenzie, C. R., Hirama, T., Brousseau, R., Lowary, T. L., Young, N. M., Bundle, D. R., and Narang, S. A. (1995). Basis for selection of improved carbohydrate- binding single-chain antibodies from synthetic gene libraries. Proc Natl Acad Sci USA 92:4992-4996. Deng, S. J., MacKenzie, C. R., Sadowska, J., Michniewicz, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1994). Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. JBiol Chem 269:9533-9538. Desmyter, A., Transue, T. R, Ghahroudi, M. A., Thi, M. H., Poortmans, F., Hamers, R., Muyldermans, S., and Wyns, L. (1996). Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme [see comments]. Nat Struct Biol 3:803-811. Hamers, C. C, Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C, Songa, E. B., Bendahman, N., and Hamers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature 363:446-448. Harrison, J. L., Williams, S. C, Winter, G., and Νissim, A. (1996). Screening of phage antibody libraries. Methods Enzymol 267:83-109. Johnson, B. A. andBlevins, R. A., J. Chem., Phys., 29, 1012-1014, 1994. Jδnsson, U., Fagerstam, L., Ivarsson, B., Johnsson, B., Karlsson, R, Lundh, K., Lδfas, S., Persson, B., Roos, H., Rόnnberg, I., Sjδlander, S., Stenberg, E., Stahlberg, R., Urbaniczky, C, Ostlin, H., and Malmqvist, M. (1991). Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 11, 620-627. Knappik, A. and Pluckthun, A. (1994). An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. BioTechniques 17:754- 761. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lauwereys, M., Arbabi, G. M., Desmyter, A., Kinne, J., Holzer, W., De Genst, E., Wyns, L., and Muyldermans, S. (1998). Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBOJ 17:3512-3520. Lowman, H. B., Bass, S. H., Simpson, N., and Wells, J. A. (1991). Selecting Mgh-affinity binding proteins by monovalent phage display. Biochemistry 30:10832-10838. MacKenzie, R. and To, R. (1998). The role of valency in the selection of anti-carbohydrate single-chain Fvs from phage display libraries. J. Immunol. Methods 220, 39-49. MacKenzie, C. R., Sharma, V., Brummell, D., Bilous, D., Dubuc, G., Sadowska, J., Young, N. M., Bundle, D. R., and Narang, S. A. (1994). Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs. Biotechnology N Y 12:390-395. MacKenzie, C.R., Hirama, T., Deng, S.-J., Bundle, D.R., Narang, S.A., and Young. N.M. (1996). Analysis by surface plasmon resonance of the influence of valence on the ligand- binding affinity and kinetics of an anti-carbohydrate antibody. J. Biol. Chem. 271, 1527-1533 McAfferty, J., Griffiths, A.D., Winter, G. and Chiswell, D.J. (1990). Nature 348, 552-554. Messing, J. (1983). New M13 vectors for cloning. Methods in Enzymology 101, 20-78. Miceli, R. M., Degraaf, M. E., and Fischer, H. D. (1994). Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope. J Immunol Methods 167:279-287. Morea, V., Tramontano, A., Rustici, M., Chothia, C, and Lesk, A. M. (1998). Conformations of the third hypervariable region in the NH domain of immunoglobulins. JMol Biol 275:269- 294. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J. A., and Hamers, R. (1994). Sequence and structure of NH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7: 1129-1135. Narang, S. A., Yao, F. L., MichnieWics, J. J., Dubuc, G., Phipps, J., and Somorjai, R. L. (1987) Protein Eng. 1, 481-485. Narang, S. A., Brummell, D. A., Sharma, V., Dubuc, G., MacKenzie, C. R., MichnieWics, J. J., Sadowska, J., Anand, N., Young, N. M., and Bundle, D.R. (1991) Nucleic Acids Res. Symp. Ser. 24, 173-179. Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C, Lane, D., and Winter, G. (1994). Antibody fragments from a 'single pot' phage display library as immunochemical reagents. EMBOJ 13:692-698. Padlan, E. A. (1994). Anatomy of the antibody molecule. Mol Immunol 31:169-217 ' . Piotto, M., Saudek, V., and Sklenar, V. (1992) J. Biomol. NMR 2, 661-665. Reiter, Y., Schuck, P., Boyd, L. F., and Plaksin, D. (1999). An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain NH molecules with a unique interface. JMol Biol 290:685-698. Sambrook, J. Fritsch E. F. and Maniatis T (1989). "Molecular Cloning: A laboratory Manual (2 nd ed.)," Cold Spring Harbor Laboratory, Cold Spring Harbor, ΝY. Sanger, F., Νicklen, S., and Coulson, A. R (1977). DΝA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467. Sheriff, S. and Constantine, K. L. (1996). Redefining the minimal antigen-binding fragment [news; comment]. Nat Struct Biol 3:733-736. Sklenar, N., Piotto, M., Leppik, R., and Saudek, V. (1993) J. Magn. Reson. A102, 241-245. Slatter, M., Schleucher, J. and Griesinger, C, Progr. ΝMR Spectrosc, 1999, 34, 93-158. Spinelli, S., Frenken, L., Bourgeois, D., de Ron, L., Bos, W., Verrips, T., Anguille, C, Cambillau, C, and Tegoni, M. (1996). The crystal structure of a llama heavy chain variable domain [letter] [see comments]. Nat Struct Biol 3:752-757. Tung, W. L. and Chow, K. C. (1995). A modified medium for efficient elecfrotransformation of E. coli. Trends Genet 11:128-129. Nieπa, O. and Messing, J. (1982). The pUC plasmids , an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268. Vu, K. B., Ghahroudi, M. A., Wyns, L., and Muyldermans, S. (1997). Comparison of llama VH sequences from conventional and heavy chain antibodies. Mol Immunol 34:1121-1131. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli [see comments]. Nature 341:544-546. Wishart, D. S. and Sykes, B. D., J Biomol NMR, 4, 171-180, 1994. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L. and Sykes, B. D., J. Biomol. NMR, 6 135-140, 1995. Zacher, A.N., Stock, C.A., Golden, J.W. and Smith, G.P. (1980). Gene 9, 127-140.

Claims: CLAIMS We claim: 1. A library for expression of immunoglobulin heavy chain domains, said library comprising a repertoire of nucleic acid sequences each encoding a polypeptide comprising a N H , said N H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-Ν, 93-A and 108-Q, said repertoire comprising a diversity of sequences which differ from one another at least in a subsequence coding for part of the CDR so as to provide nucleic acid encoding a repertoire of polypeptides comprising Nπs differing at least in said CDR and comprising at least one amino acid of said group. 2. A library according to claim 1, wherein said group further comprises 74- A, 83-K, 84- P and wherein said repertoire of nucleic acid sequences each encode a polypeptide comprising a N H which comprises any one, two, three, four, five, six, seven or eight amino acids of said group and include at least one amino acid of the group as defined in claim 1. 3. A library according to claim 1 or 2, wherein said CDR is a CDR3. 4. A library according to claim 1, 2 or 3, wherein said CDRs are derived from a preexisting repertoire of CDRs; 5. A library according to claim 1, 2 or 3, wherein said CDRs comprise random sequences. 6. A library according to claim 1, 2 or 3, wherein said nucleic acid encoding a repertoire of immunoglobulin heavy chain variable domains further comprises a subsequence encoding one or more constant domains for expression of Ig-type chains. 7. A method for generating a N H expression library having a diversity of CDR sequences, said method comprising: (a) providing expression vectors, said vectors comprising a nucleic acid sequence which encodes a N H comprising a CDR and any one, two, three or four, or five, amino acids from a " group comprising 6-A, 23-A, 82a-Ν, 93-A and 108-Q; (b) introducing by mutagenesis a variation in the said N H nucleic acid sequence; and (c) recovering an expression library having a diversity of binding activities. 8. A library which expresses N H s, said library comprising a set of framework regions carrying a diversity of CDR sequences, said library having a diversity of binding activities, said frame work regions comprising one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-Ν, 93-A and 108-Q. 9. A method according to claim 7 or a method according to claim 8, wherein said CDR is a CDR3. 10. A library according to claim 8, which expresses V H S having CDR diversity in only the CDR3 sequences. 11. A library produced by the method of claim 7. 12. A library according to claim 6, wherein said framework regions collectively are at least 90% homologous to the framework regions depicted in Figure 2. 13. A library according to claim 8 or 12 having CDRs which collectively are at least 80% homologous to the CDRs depicted in Figure 2. 14. A library according to claim 8, 12 or 13, wherein said framework regions comprise at least 2 amino acids of a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q. 15. A library according to claim 8, 12 or 13, wherein said framework regions comprise at least 3 amino acids of a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q. 16. A library according to claim 8, 12 or 13, wherein said framework regions comprise least 4 amino acids of a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q. 17. A library according to claim 8, 12 or 13, wherein said framework regions comprise 5 amino acids of a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q. 18. A polypeptide comprising a V H , said polypeptide derived from a library according to any of claims 1 to 6, 8, or 10-17. 19. A combinatorial library comprising variants of a parental V H ligand binding molecule, wherein said parental ligand binding molecule comprises an immunoglobulin V H fragment comprising at least in substantial part, at least the FR regions of the immunoglobulin V H domain depicted in Figures 2 and wherein said variants comprise, at least in substantial part, at least the FR regions of the immunoglobulin V H domain depicted in Figure 2 including at least one of amino acid residues 6, 23, 82a, 93 and 108 depicted in Figure 2, and differ from said parental ligand binding molecule in amino acid residues constituting part of at least one of the CDRs of said parental ligand binding molecule. 20. A library according to claim 19, wherein said variants comprise each of amino acid residues 6, 23, 82a, 93 and 108 depicted in Figure 2 subject only to conservative substitations which on the whole do not adversely affect the solubility properties of such variants. 21. A library according to claim 19, having a substantial representation of variants which have, a CDR3 that is 16 to 33 amino acids in length. 22. A library according to claim 21, wherein substantially all of said variants have a CDR3 that is the same length. 23. A library according to claim 21, wherein said variants have CDR3s which vary in length. 24. A library according to claim 22 or 23, predominantly comprising variants which have a CDR3 that is 18 to 28 amino acids in length. 25. A library according to claim 22 or 23, predominantly comprising variants which have a CDR3 that is 20 to 25 amino acids in length. 26. A library according to claim 22, predominantly comprising variants which have a CDR3 that is 23 amino acids in length. 27. A library according to claim 22 or 23, wherein said variants vary from said parental ligand binding molecule in amino acids constituting at least part of the CDR3. 28. A library according to claim 27, wherein said parental ligand binding molecule comprises an immunoglobulin N H binding fragment comprising, at least in substantial part, the CDR3 region of the immunoglobulin Nπ domain depicted in Figure 2. 29. A library according to claim 22, wherein said parental ligand binding molecule comprises an immunoglobulin N H binding fragment comprising, at least in substantial part, the CDR regions of the immunoglobulin V H domain depicted in Figure 2. 30. A library according to claim 22, wherein said variants comprise the same FR regions as said parental binding molecule. ι 31. A phage display library according to claim 22, wherein said parental ligand binding molecule comprises the entire FR regions of the immunoglobulin V H domain depicted in Figures 2. 32. A library according to claim 19, wherein said parental ligand binding molecule comprises at least in substantial part the FR2 region of the immunoglobulin VH domain depicted in Figure 2, including residues 44, 45 and 47 depicted in Figure 2, and wherein the FR2 regions is at least partially randomized to generate variants having one or more hydrophilic amino acids at V H -VL interface. 33. A library according to claim 20, wherein said variants vary from said parental ligand binding molecule at amino acids which are proximal to the carboxy terminus of the CDR3. 34. A library according to claim 20, wherein said variants vary from said parental ligand binding molecule in an amino acids which are immediately upstream of position lOOo. 35. A library according to claim 20, wherein said variants vary from said parental ligand binding molecule in an amino acids lOOi to lOOn identified Figure 2. 36. A library according to claim 20, wherein said parental ligand binding molecule is derived from a human V H domain identified in Figure 1 or is built on any framework which is at least 80% homologous to the framework and other conserved regions of said human V H domain. 37. A method of identifying a polypeptide comprising a V H which binds to a target ligand, comprising the steps of: (a) screening polypeptide members of the library of any one of claims 1-6, 10-17, or 19-36 for their ability to preferentially bind to the target ligand; and (b) identifying at least one polypeptide member which binds to the target ligand. 38. A library as claimed in any one of claims 1-6, 10-17, or 19-36, wherein said Vπis human VH. 39. An immunoglobulin domain comprising a polypeptide comprising a V H , said V H comprising a CDR and any one, two, three or four, or five, amino acids from a group comprising 6-A, 23-A, 82a-N, 93-A and 108-Q. 40. An immunoglobulin domain according to claim 38, wherein said group further comprises 74-A, 83-K, 84-P. 41. An immunoglobulin domain according to claim 38 or 39, wherein said CDR is a CDR3. 42. An immunoglobulin domain according to claim 38, 39 or 40, wherein said CDR is derived from a pre-existing repertoire of CDRs. 43. An immunoglobulin domain according to claim 38, 39 or 40, wherein said CDR comprises random sequences. '44. An immunoglobulin domain according to claim 38, 39 or 40, wherein said immunoglobulin heavy chain variable domains further comprises one or more constant domains for expression of Ig-type chains. 45. An immunoglobulin domain comprising a variant of a parental V H ligand binding molecule, wherein said parental ligand binding molecule comprises an immunoglobulin V H fragment comprising at least in substantial part, at least the FR regions of the immunoglobulin V H domain depicted in Figure 2 and wherein said variants comprise, at least in substantial part, at least the FR regions of the immunoglobulin V H domain depicted in Figure 2 including at least one of amino acid residues 6, 23, 82a, 93 and 108 depicted in Figure 2, and differ from said parental ligand binding molecule in amino acid residues constituting part of at least one of the CDRs of said parental ligand binding molecule. 46. An immunoglobulin domain according to claim 45, wherein said variant comprises each of amino acid residues 6, 23, 82a, 93 and 108 depicted in Figure 2 subject only to conservative substitations which on the whole do not adversely affect the solubility properties of said variant. 47. An immunoglobulin domain according to claim 45, having a CDR3 that is 16 to 33 amino acids in length. 48. An immunoglobulin domain according to claim 47, having a CDR3 that is 18 to 28 amino acids in length. 49. An immunoglobulin domain according to claim 48, having a CDR3 that is 20 to 25 amino acids in length. 50. An immunoglobulin domain according to claim 49, having a CDR3 that is 23 amino acids in length. 51. An immunoglobulin domain according to claim 45, wherein said variants vary from said parental ligand binding molecule in amino acids constituting at least part of the CDR3. 52. An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule comprises an immunoglobulin VH binding fragment comprising, at least in substantial part, the CDR3 region of the immunoglobulin VH domain depicted in Figure 2. 53. An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule comprises an immunoglobulin VH binding fragment comprising, at least in substantial part, the CDR regions of the immunoglobulin VH domain depicted in Figure 2. 54. An immunoglobulin domain according to claim 45, having the same FR regions as said parental binding molecule. 55. An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule comprises the entire FR regions of the immunoglobulin VH domain depicted in Figures 2. 56. An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule comprises at least in substantial part the FR2 region of the immunoglobulin VH domain depicted in Figure 2, including residues 44, 45 and 47 depicted in Figure 2, and wherein the FR2 regions is at least partially randomized to generate variants having one or more hydrophilic amino acids at VH-VL interface. 57. An immunoglobulin domain according to claim 45, wherein said variants vary from said parental ligand binding molecule at amino acids which are proximal to the carboxy terminus of the CDR3. 58. An immunoglobulin domain according to claim 45, wherein said variants vary from said parental ligand binding molecule in amino acids which are immediately upstream of position lOOo. 59. An immunoglobulin domain according to claim 45, wherein said variants vary from said parental ligand binding molecule in amino acids lOOi to lOOn identified Figure 2. 60. An immunoglobulin domain according to claim 45, wherein said parental ligand binding molecule is derived from a human VH domain identified in Figure 1 or is built on any framework which is at least 80% homologous to the framework and other conserved regions of said human VH domain. 61. An immunoglobulin domain as claimed in any one of claims 39-60, wherein said V H is human V H .

Inventors: DAN MICHAEL, ENTWISTLE JOCELYN, MACKENZIE COLIN R, NARANG SARAN, TANHA JAMSHID, DAN, MICHAEL, ENTWISTLE, JOCELYN, MACKENZIE, COLIN, R., NARANG, SARAN, TANHA, JAMSHID
Applicants: DAN MICHAEL, ENTWISTLE JOCELYN, GRAD CAROLE, MACKENZIE COLIN R, NARANG SARAN, TANHA JAMSHID, DAN, MICHAEL, ENTWISTLE, JOCELYN, GRAD, CAROLE, MACKENZIE, COLIN, R., NARANG, SARAN, TANHA, JAMSHID
Application year: 2001
Main classification: C07K16
IPC codes: C07K16, C12N15, C40B40