MXPA06000080A - Look-through mutagenesis - Google Patents

Abstract

A method of mutagenesis by which a predetermined amino acid is introduced into each and every position of a selected set of positions in a preselected region (or several different regions) of a polypeptide to produce a library of polypeptide analogs. The method is based on the premise that certain amino acids play crucial role in the structure and function of proteins. Libraries can be generated which contain only desired polypeptide analogs and are of reasonable size for screening. The libraries can be used to study the role of specific amino acids in polypeptide structure and function and to develop new or improved polypeptides such as antibodies, antibody fragments, single chain antibodies, enzymes, and ligands.

Description

UTAGENESIS BY REVISION Related Requests and Information This application claims priority to United States Provisional Application No. 60/483282, filed on June 27, 2003, the entire contents of which are incorporated herein by reference. The entire contents of all other patents, patent applications, and references cited throughout the following specification are also incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION Mutagenesis is a useful tool in the study and function of protein structure. Mutagenesis can be performed on the nucleotide sequence of a cloned gene that encodes a protein of interest and the modified gene can be expressed to produce mutants of the protein. By comparing the properties of the wild-type protein and the mutants generated, it is often possible to identify individual amino acids or amino acid domains that are essential for the structural integrity and / or biochemical function of the protein, such as its binding and / or catalytic activity. . However, the number of mutants that can be generated from the individual protein makes it difficult to select mutants that will be informative or have the desired property, even if the selected mutants encompassing the mutations are only in important putative regions of a protein (eg. example, regions that make up an active site of a protein). For example, the substitution, deletion, or insertion of a particular amino acid may have a local or global effect on the protein. The above procedures for mutagenizing polypeptides have been either too restrictive, too much, or limited to inactivate the function of the proteins rather than gain or improve function. For example, a highly restrictive approach is selective or site-directed mutagenesis that is used to identify the presence of a particular functional site or understand the sequences of realization of a highly specified alteration within the functional site. A common application of site-directed mutagenesis in the study of phosphoproteins in which an amino acid residue, which ordinarily would phosphorylate and allow the polypeptide to carry out its function, is altered to confirm the binding between phosphorylation and functional activity. This approach is very specific for the polypeptide and rest that is being studied. Reciprocally, a highly inclusive approach is saturation or random mutagenesis that is designed to produce a large number of mutations that encompass all possible alterations within a defined region of a gene or protein. This is based on the principle that, by generating essentially all possible variants of a relevant protein domain, the appropriate arrangement of amino acids to be produced as one of the randomly generated mutants. However, in practice, the vast number of random combinations of mutations generated can prevent the ability to significantly select a desired candidate due to the presence of so-called "noise" in this way many unwanted candidates. Another approach, termed "wait" mutagenesis (see patents Nos. 5,830,650; 5,798,208) has been used to mutagenize a defined region of a polypeptide by synthesizing a mezcal of degenerate oligonucleotides that, statistically, contain a desired set of mutations. However, due to the synthesis of degenerate polynucleotides, walking mutagenesis produces a number of undesired alterations in addition to the desired set of mutations. For example, to sequentially introduce a mutation through a defined region of five amino acid positions only, a set of about 100 polynucleotides can be prepared (and selected) (see, for example, Figure 6). According with the last, to prepare and select, for example, two or three regions that become growing complex, that is, that requires the preparation and selection of 200 to up to 300 polynucleotides, respectively, for the presence of 10 to 15 mutations only. Still in another approach that has been used to mutagenize proteins is the alanine scanning mutagenesis, in which an alanine residue is "scanned" through a portion of a protein to identify positions in which the function of proteins is interrupted. However, this approach only looks at the loss of protein function by replacing a neutral alanine residue at a given position, rather than gaining or improving function. In this way, it is not a useful approach to generate proteins that have improved structure and function. According to the above, there remains a need for a systematic way to mutagenize a protein for a new or improved function.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to a method of mutagenesis for the generation of novel or improved proteins (or polypeptides) and to libraries of analogs of specific polypeptides and polypeptides generated by the processes. The polypeptide targeted for mutagenesis can be a natural, synthetic or engineered polypeptide, including fragments, analogs and the mutant forms thereof. In one embodiment, the method comprises introducing a predetermined amino acid essentially at each position within a defined region (or several different regions) of the amino acid sequence of a polypeptide. A library of polypeptides is generated that contains analogs of polypeptides that individually do not individually have more than one predetermined amino acid, but that collectively have the predetermined amino acid at each position within the defined region (s). The process can be termed a "revision" of mutagenesis because, in effect, a predetermined individual amino acid (and only the predetermined amino acid) is replaced position by position along a more defined region (s) (s). ) of a polypeptide. In this way the invention allows to "revise" the structural and functional consequences of substitution separately from a predetermined amino acid at each amino acid position within a defined region of the polypeptide thereby segregating a specific protein chemistry to the defined region without no interference or "noise" from the generation of undesired polypeptide analogs (ie analogues containing amino acid substitutions other than those following the "revision" scheme) see for example Figure 6. Accordingly, the present invention allows the highly efficient and accurate systematic evaluation of the role of a specific amino acid change in one or more defined regions of a polypeptide. This becomes particularly important when evaluating (by mutation) two or more defined regions, so that the number of polypeptide analogs required is greatly increased and, thus, the presence of undesired analogues also increases. . The present invention obviates this problem by the complete elimination of undesired analogs and, thus, the potential that any changes in protein structure or function observed are the result of anything but the substitution of the predetermined amino acid. In this way, the effect of segregating a specific protein chemistry to even multiple regions with a protein can be studied with high precision and efficiency. Importantly, this includes the study of how mutagenesis can effect the interaction of such regions, thereby improving the overall structure and function of the protein. In a particular embodiment of the invention, the library of polypeptide analogs is generated and selected by first synthesizing the individual polynucleotides that encode a region or regions of a polypeptide in which, collectively, the polynucleotides represent all possible polynucleotides. according to the review criteria described in this specification. Variant polynucleotides are expressed, for example, using in vitro transcription and translation and / or using a representation technology, such as ribosome representation, phage display, bacterial representation, yeast representation, serial representation or any other representation system suitable known in the art. The expressed polypeptides are then screened and selected using functional assays, such as binding assays or enzymatic / catalytic assays. In one embodiment, the polypeptides are expressed in association with the polynucleotide encoding the polypeptide, thereby allowing identification of the polynucleotide sequence encoding the polypeptide. Still in another modality, polypeptides are synthesized directly using protein chemistry. Thus, the present invention provides a mutagenesis method that can be used to generate libraries of polypeptide analogues that are of a practical size for selection, in part, because the libraries are devoid of undesired analogue polypeptide or also called noise. The method can be used to study the role of specific amino acids in the structure and function of polypeptides and to develop new or improved polypeptides such as antibodies, binding fragments or analogs thereof, individual chain antibodies, catalytic antibodies, enzymes and ligands. . In addition, the method can be performed with the benefit of a priori information, for example, by computer modeling, which can be used to select an initial subset of polypeptide analogs to be produced and studied using "revision" mutagenesis. Other advantages and aspects of the present invention will be readily apparent from the following description and examples.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the exemplary defined regions (or D regions) that can be examined using Revision Mutagenesis (LTM) and functional assays to identify analogues of desired polypeptides from such D regions.

Figure 2 illustrates the use of LTMs between defined regions in an antibody variable region (ie, CDR1, CDR2, and CDR3 of an antibody heavy chain variable region). The variable region of the light chain can be screened similarly, either alone or in combination with the variable region of the heavy chain. For convenience in subsequent screening assays, the variable region of the heavy chain can be screened in the context of an individual chain antibody (sFv) as shown. Figure 3 illustrates the use of LTM in a defined region (i.e., positions 31-25 of CDR1) of a variable region of the heavy chain. Figure 4 illustrates the use of LTM in a defined region (i.e., positions 55-68 of CDR2) of a variable region of the heavy chain. Figure 5 illustrates the use of LTM in a defined region (i.e., positions 101-111 of CDR3) of a variable region of the heavy chain. Figure 6 illustrates the advantages of LTM compared to standby mutagenesis. The LTM of a defined representative region, ie CDR1 of an antibody heavy chain variable region, results in the sequential alteration of each amino acid position by the defined region, without the introduction of any unwanted amino acid residue or also called noise. Figure 7 illustrates the integration of three defined regions of a protein again in the context of the proteins following revision mutagenesis, in particular, the integration of the three CDRs of a variable region of the heavy chain into an antibody format of a single chain following the mutagenesis by revision. Figure 8 illustrates the use of the polymerase chain reaction (PCR) to construct defined regions of a heavy and light chain of antibody subjected to LTM in a larger gene context. Figure 9 illustrates in exemplary fashion the diversity of formats of each of the CDRs in a variable region of antibody to obtain a cartalitic site comprising, for example, a serine, histidine, and / or aspartic acid and as may be available . Figure 10 illustrates the integration of the six CDRs of a binding region of an antibody that has been subjected to LTM and the resulting diversity if a predetermined amino acid residue of twenty different predetermined amino acid residues is used. Figure 11 illustrates the integration of the six CDRs of an individual anti-TNF chain antibody (sFv) and that has been subjected to LTM and the resulting diversity if a predetermined amino acid residue of three different predetermined amino acid residues is used. Figure 12 illustrates an ordered library representing some of the possible polypeptide analogs of the six CDRs of an antibody binding region that can be achieved using LTM. Figure 13 illustrates the selection of an ordered expression library using free ribosome representation. Figure 14 illustrates the combinatorial chemistry explored when the binding region of an antibody variable region (ie, all six CDRs) is subjected to LTM. Figure 15 shows the sequence of the variable region (in single chain format) of several representative anti-TNF binding molecules that are subject to LTM. Figure 16 shows a scheme for carrying out the protease selection assay for catalytic LTM selection candidates. Figure 17 shows a scheme detailing the mechanism (and advantages) of the protease selection assay when carried out in bacterial cells. Figure 18 shows a flow chart detailing the mechanics of gene selection libraries for catalytic activity, eg, catalytic antibody activity, using either ribosome or yeast representation. Figure 19 shows a scheme for carrying out LTM when one can coordinate information (for example, computer modeling information) and empirical information (test results) for more efficient molecule design and development. This guided LTM approach is called "through guide" mutagenesis. Figure 20 shows a hypothetical wild type sequence VH CDR3 (shaded ovals in the upper part) and the resulting sequences in the library members of the His substitution by LTM (in blank ovals). Individual LTM His substitutions are encoded by individual oligonucleotides, for example, oligonucleotides synthesized in a high throughput manner. The subset libraries of LTM for the other amino acid substitutions (LTM) in this CDR domain are constructed in a similar manner. Figure 21 illustrates the generation of scFv libraries. In the line of the upper part of the x-axis and the column far from the left part of the x axis, the three digits represent the 3 CDRs on each of the light and heavy chains. A "0" indicates a wild-type CDR sequence, while a "1" indicates a CDR mutated by LTM. The number on the grid indicates the complexity of the subset library. For example, in the upper left corner of the matrix it is a "0" when the corresponding x and y axes are "000" and "000" indicating that none of the CDRs in either the VH and VL are respectively mutated. Moving a row from the "0" end to the position of the neighboring grid "1" would be designed by the axis "100" and "000" on the y-axis indicating that VH CDR1 is mutated while V CDR remains wild type. In this way, similarly, a grid numbering of "4" would mean that there is a grid numbering and "4" would mean that there are four CDRs mutated simultaneously. Initially the seven VH and VL chains (indicated by the arrows) are prepared using SOE-PCR. The VH and VL chains are then amplified and mixed and matched by a mega primer to generate the remaining VH and VL combinations in one step.

DETAILED DESCRIPTION OF THE INVENTION In order to prevent a clear understanding of the specification and claims, the following definitions are provided below.

Definitions As used herein, the term "analog" refers to a variant or polypeptide polypeptide (or a nucleic acid encoding such polypeptide) having one or more amino acid substitutions. The term "binding molecule" refers to any binding molecule, including proteins, polypeptides, peptides, and small molecules, which bind to a substrate or target. In one embodiment, the binding molecule is an antibody or binding fragment (e.g., a Frab fragment), single domain antibody, single chain antibody (e.g., sFv), or peptide capable of binding to a ligand. The term "defined region" refers to a selected region of a polypeptide. Typically, the defined region includes all or a portion of a functional site, for example, the binding site of a ligand, the binding site of a binding or receptor molecule, or a catalytic site. The defined region may also include multiple portions of a functional site. For example, the defined region can include all, or multiple portions of, a complementary heavy determining region (CDR) or a heavy and / or light chain variable region (VR) of an antibody. In this way, the functional site can include a defined single or multiple region that contributes to the functional activity of the molecule. The term "library" refers to two or more molecules mutagenized according to the method of the invention. The library molecules may be in the form of polynucleotides, polypeptides, polynucleotides and polypeptides, polynucleotides and polypeptides in a cell-free extract, or as polynucleotides and / or polypeptides in the context of a phage, prokaryotic cells, or in eukaryotic cells . The term "mutagenization" refers to the alteration of an amino acid sequence. This can be achieved by altering or producing a nucleic acid (polynucleotide) capable of encoding the altered amino acid sequence, or by direct synthesis of an altered polypeptide using protein chemistry. The term "polynucleotide (s)" refers to nucleic acids such as DNA molecules and RNA molecules and analogs thereof (for example), DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry). As desired, the polynucleotides can be prepared synthetically, for example, using art-recognized nucleic acid chemistry or enzymatically using, for example, a polymerase. Typical modifications include mutilation, biotinylation, and other modifications known in the art. In addition, the nucleic acid molecule can be single chain or double chain, when desired, linked or associated (eg, covalently or non-covalently) to a detectable moiety. The term "variant polynucleotide" refers to a polynucleotide that encodes a corresponding polypeptide analogue (or portion thereof) of the invention. Thus, variant polynucleotides contain one or more codons that have been changed to result in the expression of a different amino acid. The term "polypeptide (s)" refers to two or more amino acids linked by a peptide bond, for example, peptides (eg, from about 50 amino acid residues), as well as longer peptide sequences, eg, protein deceases. which typically comprise amino acid sequences from how little amino acid residues to more than 1000 amino acid residues. The term "combination" refers to the combination of polypeptide or polypeptide analog variants to form libraries that represent the mutagenesis by screening of an entire polypeptide region. The molecules can be in the form of a polynucleotide and / or polypeptide and can coexist in the form of a sub-library, as molecules of a solid support, as molecules in solution, and / or as molecules in one or more organisms (eg, phage, prokaryotic cells, or eukaryotic cells). The term "predetermined amino acid" refers to an amino acid residue selected for substitution at each position within a defined region or polypeptide to be mutagenized. This does not include position (s) within the region that already (for example, naturally) contains the predetermined amino acid and, thus, needs to be substituted with the predetermined amino acid. Accordingly, each polypeptide analogue generated according to the present invention contains no more than one "predetermined amino acid" residue in a given given region. However, collectively, the library of generated protein analogs contains the predetermined amino acid in each position of the region being mutagenized. Typically, a predetermined amino acid is selected for a particular or chemical size usually associated with the side group of the amino acid. Suitable predetermined amino acids include, for example, glycine and alanine (stereastically small); serine, threonine, and cysteine (nucleophilic); valine, leucine, isoleucine, methionine, and proline (hydrophobic); phenylalanine, tyrosine, and tryptophan (aromatic); aspartate and glutamate (acid); asparagine, glutamine, and histidine (amide); and lysine and arginine (basic).

PREFERRED MODALITY OF THE INVENTION The study of proteins has revealed that certain amino acids play a crucial role in their structure and function. For example, it appears that only a number of amino acids participate in the binding of an antibody to an antigen or are involved in the catalytic case of an enzyme. Although it is clear that certain amino acids are critical for the activity or function of proteins, it is difficult to identify which amino acids are involved, how they are involved, and what substitutions can improve protein structure or function. In part, this is due to the complexity of the configuration spacing amino acid side chains into polypeptides and the interrelation of different portions of the polypeptide that contributes to forming a functional site. For example, the interrelation between the six CDRs of the variable regions of heavy and light chains of an antibody contributes to the pocket of binding to antigen or ligand. Prior mutagenesis procedures, such as selective (site-directed) mutagenesis and saturation mutagenesis, are of limited utility for the study of protein structure and function in view of the enormous number of possible variants in complex polypeptides. This is especially true since desirable combinations are often accompanied by the presence of vast amounts of undesirable combinations or also called noise. The method of this invention provides a systematic, practical, and highly accurate approach to evaluating the role of particular amino acids and their position, within a defined region of a polypeptide, in the structure or function of the polypeptide and, thus, to produce improved polypeptides. 1. Selection of a defined region According to the present invention, a defined region or regions within a protein are selected by mutagenesis. Typically, the regions are believed to be important for the structure or function of the proteins (see, for example, Figure 1). This can be deduced, for example, from which the structural and functional aspects are known or can be deduced from the comparison of the defined region (s) that are known from the study of other proteins, and can help by modeling the information. For example, the defined region may be one that has a role in a functional site, for example, in binding catalysis, or another function. In one embodiment, the finite region is a hypervariable region or complementarity determining region (CDR) of an antigen-binding molecule. In another embodiment, the defined region is a portion of a complementarity determining region (CDR). In another embodiment, two or more defined regions, eg, CDRs or portions thereof, are selected for mutagenesis. 2. Selection of a predetermined amino acid residue The rest of the amino acids chosen for substitution within the defined 8s) region (s) is generally selected from those known to be involved in the structure or function of interest. The twenty amino acids of natural origin differ with respect to their secondary chain. Each secondary chain is responsible for chemical properties that make each amino acid unique. For the purpose of altering the binding or creating new binding affinities, any of the naturally occurring amino acids can be selected in general. Thus, previous mutagenesis procedures, which create vast numbers of analogs for each substitution, were not practical to evaluate the effect on substitution protein binding of each of the twenty amino acids. Conversely, the methods of the present invention create a practical number of analogs for each of the amino acid substitutions and, thus, allow the evaluation of a variety of protein chemistries within a region or regions of a protein. In contrast to protein binding, only a subset of amino acid residues typically participate in non-catalytic enzyme events. For example, from the chemical properties of the side chains, only a selected number of natural amino acids preferentially participate in catalytic episodes. These amino acids belong to the group of polar and neutral amino acids such as Ser, Thr, Gln, Tyr, and Cys, the group of charged amino acids, Asp and Glu, Lys and Arg, and especially the His amino acid. Other polar and neutral side chains are those of Cys, Ser, Thr, Asn, Gln and Tyr. Gly is also considered to be a limiting member of this group. Ser and Thr play an important role in the formation of hydrogen bonds. Thr has an additional asymmetry in the beta carbon, therefore only one of the stereoisomers is used. The amino acid Gln and Asn can also form hydrogen bonds, the amido groups that function as hydrogen donors and the carbonyl groups that function as acceptors. Gln has a more CH2 group than Asn that makes the polar group more flexible and reduces its interaction with the main chain. Tyr has a very polar hydroxyl group (phenolic OH) that can be dissociated at high pH values. Tyr behaves in some way like a charged secondary chain; its hydrogen bonds are quite strong. Neutral polar acids are found in nature as well as in the interior of protein molecules. As internal residues, they usually form hydrogen bonds with each other or with the main structure of the polypeptide. Cys can form disulfide bridges. Histidine (His) has a heterocyclic aromatic side chain with a pK value of 6.0. In the physiological pH range, its imidazole ring may be either uncharged or charged, after picking up a hydrogen ion from the solution. Since these two states are readily available, His is completely adequate to catalyze chemical reactions. It is found in most active centers of enzymes, for example, serine proteases. Asp and Glu are negatively charged physiological pH. Due to its short secondary chain, the carboxyl group of Asp is quite rigid with respect to the main chain. This may be the reason why the carbonyl group in many catalytic sites is provided by Asp and not by Glu. The charged acids are generally found on the surface of a polypeptide. In addition, Lys and Arg are on the surface. They have long and flexible secondary chains that have multiple rotamers or similar energies. In several cases, Lys and Arg take part in the formation of internal salt bridges or their help in catalysis. Due to its exposure to the surface of the polypeptide, Lys is a moiety most frequently recognized by enzymes that either modify the secondary chain or cleave the peptide chain at the carbonyl end of the Lys moieties. While the secondary group chemistry of an amino acid can guide the selection of a predetermined amino acid residue, the lack of a desired secondary group chemistry can be a criterion for excluding an amino acid residue for use as the predetermined amino acid. For example, sterically small and chemically neutral amino acids, such as alanine, can be excluded from mutagenesis by revision to lack a desired chemistry. 3. Synthesis of libraries of polypeptide analogs In one embodiment, a library of polypeptide analogs is generated to be selected by synthesis of individual oligonucleotides that encode the defined region of the polypeptide and have no more than one codon for the predetermined amino acid. This is accomplished by incorporating at each codon position within the oligonucleotide either the codon required for the synthesis of wild-type polypeptide or a codon for the predetermined amino acid. This differs from the oligonucleotides produced in saturation mutagenesis, random mutagenesis, or mutagenesis by revision because, for each oligonucleotide, only one mutation is made, opposite to multiple mutations. The oligonucleotides can be produced individually and then mixed or combined as desired. When the codon of the wild-type sequence and the codon for the amino acid are the same, no substitution is made. According to the above, the number of amino acid positions within the defined region will determine the maximum number of prepared oligonucleotides. For example, if five codon positions are altered with the predetermined amino acid, then five oligonucleotides plus one polynucleotide representing the wild-type amino acid sequence are synthesized. Two or more regions may be altered simultaneously. The mixture of oligonucleotides for generating the library can be easily synthesized by known methods for DNA synthesis. The preferred process involves the use of solid phase beta-cyanoethyl phosphoramidite chemistry. See U.S. Patent No. 4,725,677. For convenience, an instrument for automatic DNA synthesis containing specific nucleotide reagent containers can be used. The polynucleotides can also be synthesized to contain restriction sites or primer hybridization sites to facilitate the introduction or assembly of the polynucleotides that represent, for example, a defined region in a larger gene context. The synthesized polynucleotides can be inserted into a larger gene context of the polypeptide that is being mutagenized using conventional genetic engineering techniques. For example, polynucleotides can be prepared to contain recognition flanking sites for restriction enzymes. See, Crea, R., U.S. Patent No. 4,888,286. The recognition sites are designed to correspond to recognition sites that either exist naturally or are introduced into the gene next to the DNA encoding the region. After conversion to the double-stranded form, the polynucleotides are ligated into the gene by conventional techniques. By means of an appropriate vector (which includes, for example, phage vectors, plasmids) the genes can be introduced into an extract without cells, prokaryotic cells, or eukaryotic cells suitable for the expression of the mutant polypeptides. In cases in which the amino acid sequence of the polypeptide to be mutagenized is known or when the DNA sequence is known, the synthesis of genes in a possible approach. For example, partially overlapping polynucleotides, typically about 20-60 nucleotides in length can be designed. The internal polynucleotides are then phosphorylated to their complementary partner to provide a double stranded DNA molecule with single chain extensions useful for further hybridization. The hybridized pairs can then be mixed together and ligated to form a full-length double-stranded molecule (see, for example, Figure 8). Suitable restriction sites can be designed near the ends of the synthetic gene for cloning into a suitable vector. The full-length molecules can be cleaved with the restriction enzymes and ligated into a suitable vector. Suitable restriction sites can also be incorporated into the synthetic gene sequence to facilitate the introduction of mutagenic modules. As an alternative for the synthesis of polynucleotides that represent the full-length double-stranded gene, polynucleotides that partially overlap at their 3 'ends (ie, with complementary 3' ends) can be assembled into an open structure and then loaded with a suitable polymerase to prepare a full-length double-stranded gene. Typically, the superposition polynucleotides are between 40-90 nucleotides in length. The extended polynucleotides are then ligated. Suitable restriction sites can be introduced in Iso extremes and / or internally for cloning purposes. After digestion with an appropriate enzyme or restriction enzyme (s), the gene fragment is ligated into a suitable vector. Alternatively, the gene fragment can be ligated at the blunt end into an appropriate vector. In these approaches, if suitable restriction sites are available (naturally or genetically engineered) after gene assembly, the degenerate polynucleotides can subsequently be introduced by cloning the module into an appropriate vector. Alternatively, degenerate polynucleotides can be incorporated in the gene assembly phase. For example, when both strands of the gene are synthesized completely chemically, complementary degenerate polynucleotides can be produced. The complementary pairs will hybridize with each other. When partially overlapping polynucleotides are used in the assembly of genes, a set of degenerate nucleotides can also be incorporated directly in place of one of the polynucleotides. The appropriate complementary chain is synthesized during the extension reaction from a polynucleotide partially complementary to the other chain by enzymatic extension with a polymerase. The incorporation of the degenerate polynucleotides in the synthesis phase also simplifies cloning when more than one domain or defined region of a gene is mutagenized. In another approach, the gene of interest is present in a single chain plasmid. For example, the gene can be cloned into a phage vector or a vector with a filamentous replication phage origin that allows the propagation of single chain molecules with the use of an auxiliary phage. The single-stranded template can be hybridized to a set of degenerate polynucleotides which represent the desired mutations and elongate and ligate, thus incorporating each analogous chain into a population of molecules that can be introduced into an appropriate host (Sayers, JR et al. ., Nucleic Acids Res. 16: 791-802 (1988) This approach can avoid many stages of cloning when multiple domains are selected for mutagenesis.The polymerase chain reaction (PCR) methodology can also be used to incorporate polynucleotides in a gene For example, the polynucleotides themselves can be used as primers for extension In this approach, the polynucleotides encoding the mutagenic modules that correspond to the defined region (or portion thereof) are complementary to each other, at least in part, and can be extended to form a large gene module that uses a polymerase, for example use, using PCR amplification. The size of the library will vary depending on the length and number of regions and amino acids within a region that is mutagenized. Preferably, the library is designed to contain less than 1015, 1014, 1013, 1212, 1011, 1010, 109, 108, 107 and more preferably, 106 polypeptide analogues or less. The above description has focused on the mutagenesis of polypeptides and polypeptide libraries by altering the polynucleotide encoding the corresponding polypeptide. However, it is understood that the scope of the invention also encompasses methods of mutagenizing polypeptides by direct synthesis of the desired polypeptide analogs using protein chemistry. When this approach is carried out, the resulting polypeptides still incorporate the features of the invention except that the use of a polynucleotides is eliminated. For the libraries described above, either in the form of corresponding polynucleotides and / or polypeptides, it is understood that the libraries may also be attached to a solid support, such as a microchip, and preferably ordered, using techniques recognized in the art. 4. Expression and selection systems Polynucleotide libraries generated by any of the above techniques or other suitable techniques can be expressed and screened to identify analogs of polypeptides having the desired structure and / or activity. The expression of the polypeptide analogs can be carried out using any suitable expression representation system known in the art including, but not limited to, cell-free extract representation system (e.g., representation of ribosomes and ordered representation systems) (for example, micro-coordinates or macro-coordinates), bacterial representation systems, phage display systems, prokaryotic cells, and / or eukaryotic cells (for example, yeast representation systems) .In one embodiment, polynucleotides are modified by genetic engineering. to serve as templates that can be expressed in an extract without cells Vectors and extracts as described, for example, in U.S. Patent Nos. 5,324,637; 5,492,817; ,665,563, can be used and many are commercially available. The representation of ribosomes and other techniques without cells can be used for binding to a polynucleotide (e.g., a genotype) to a polypeptide (i.e., a phenotype) e.g. Profusion ™ (see for example, U.S. Pat. numbers 6,348,315, 621, 804, 6,258,558, and 6,214,553). Alternatively, the polynucleotides of the invention can be expressed in a convenient expression system of E. coli, such as that described by Pluckthum and Skerra. (Pluckthum, A. and Skerra, A., Meth. Enzymol., 178: 476-515 (1989); Skerra, A. et al., Biotechnology 9: 273-178 (1991)). Mutant proteins can be expressed for secretion in the medium and / or cytoplasm of the bacterium, as described by M. Better and A. Horwitz, Meth. Enzymol. 178: 476 (1989). In one embodiment, the individual domains encoding VH and VL are each attached to the 3 'end of a sequence encoding a signal sequence, such as the signal sequence ompA, phoA or pelB (Lei, SP et al., J. Bacterial 169: 4379 (1987) These gene fusions are assembled into a dicistronic construct, so that they can be expressed from an individual vector, and secreted into the periplasmic space of E. coli when they are replicated and can be recovered in an active form, (Skerra, A. et al., expressed simultaneously with the light-chain genes of antibodies that produce antibodies and antibody fragments.) Still in another embodiment, the polynucleotides can be expressed in eukaryotic cells such as the use of yeasts, for example, representation of yeasts as described for example, in U.S. Patent Nos. 6,423,538, 6,331, 391, and 6,300,065: In this approach, the polypeptide analogues of a The library is condensed to a polypeptide that is expressed and represented on the surface of the yeast. Other eukaryotic cells can also be used for the expression of the polypeptides of the invention, such as mammalian cells, for example myeloma cells, hybridoma cells, or Chinese hamster ovary (CHO) cells. Typically, polypeptide analogs when expressed in mammalian cells are designed to be expressed in the culture medium, or expressed on the surface of such a cell. The antibody or antibody fragment can be produced, for example, a whole antibody molecule or as individual VH and VL fragments, Fab fragments, individual domains, or as individual chains (sFv) (see, Huston, JS et al., Proc. Nati, Acad. Sci. USA 85: 5879-5883 (1988)). The selection of the expressed polypeptide analogues (or polypeptides produced by direct synthesis) can be made by any appropriate means. For example, the binding activity can be evaluated by conventional immunoassay and / or affinity chromatography and catalytic activity can be determined by suitable assays for the conversion of substrates. The selection of polypeptide analogs of the invention for proteolytic function can be carried out using a conventional hemoglobin plate assay as described, for example, in U.S. Patent No. 5,798,208.5. Mutagenesis by assisted revision by modeling by ordering The mutagenesis by revision of the invention can also be carried out with the benefit of structural or modeled information concerning the analogs of polypeptides to be generated, so that the potential to generate analogues having the function Improved desired increases. The structural or modeling information can also be used to guide the selection of predetermined amino acids to be introduced into the defined regions. Still further, the actual results obtained with the polypeptide analogs of the invention can guide the selection (or exclusion) of subsequent polypeptides to be prepared and selected in an interactive manner. Accordingly, the structural or modeling information can be used to generate initial subsets of polypeptide analogs for use in the invention, thereby further increasing the efficiency of generation of improved polypeptides. In a particular embodiment, silico modeling is used to eliminate the production of any predicted polypeptide analogue having poor or undesired structure and / or function. In this way, the number of polypeptide analogs to be produced can be markedly reduced thereby increasing the signal to noise in subsequent selection trials. In another particular embodiment, silico modeling is continually updated with additional modeling, from any relevant source, for example, from a gene and protein sequence and the three-dimensional database and / or analogous results. previously tested, so that the silico database becomes more accurate in its predictive capacity (figure 19). In yet another embodiment, the silico database is provided with the test results of the previously tested polypeptide analogs and classifies the analogues, based on the criterion or test criteria, as effectors or non-effectors, eg, as analogues. of polypeptides that bind well or not so well or by being enzymatic / catalytic or not being as enzymatic / catalytic. In this way the mutagenesis by guiding the invention can match a range of functional response with particular structural information and be used as information to guide the production of analogues of future polypeptides to be tested. Accordingly, the method is especially suitable for selecting antibodies or antibody fragments for a particular function, such as binding affinity (e.g., specificity), stability (e.g., half-life) and / or effector function (e.g. , activation of complements and ADCC). Accordingly, mutagenesis of non-contiguous residues within a region may be desirable if it is known, for example, by silico modeling that certain residues in the region will not participate in the desired function. The coordinate structure and spatial interrelation between the desired regions, for example, the functional amino acid residues in the defined regions of the polypeptide, for example, the predetermined amino acid (s) that have been introduced, can be considered and modeled . Such modeling criteria, for example, chemistry of secondary groups of amino acids, atomic distances, crystallography data, etc. According to residues of the above, the number of polypeptide analogs to be produced can be minimized intelligently. In a preferred embodiment, one or more of the above steps are computer assisted. The method is also capable of being carried out, in part or in whole, by a computer-directed device. In accordance with the foregoing, the instructions for carrying out the procedure, in part or in whole, can be conferred to a means suitable for use in an electronic device to carry out the instructions. In total, the methods of the invention are capable of a high performance approach comprising software (for example, computer readable instructions) and hardware (for example, computers, robots and chips). 6. Exploration of the combinatorial chemistry of defined multiple regions The present invention provides the important advantage of allowing the evaluation by mutagenesis of several different regions or domains of a polypeptide simultaneously. This can be done using the same or different predetermined different amino acid within each region, allowing the evaluation of amino acid substitutions in conformationally related regions, so that regions that after folding of the polypeptide are associated to prepare a functional site (for example, the binding site of an antibody or the catalytic site of an enzyme). This, in turn, provides an effective way to create new or improved functional sites. For example, as shown in Figure 14, the six CDRs of an antibody that prepare the unique aspects of the antigen binding site (Fv region), can be mutagenized simultaneously, or separately in the VH or VL chains, to study the Three-dimensional interrelationships of selected amino acids in this site. In one embodiment, the combinatorial chemistry of the three or more regions are screened systematically using revision mutagenesis, and preferably six defined regions, for example the six CDRs of a variable region of the antibody heavy and light chain. To perform this mutagenesis by review on a CDR, typically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, or more amino acid positions are altered. In accordance with the foregoing, the present invention opens up new possibilities for the design of very different types of new and improved polypeptides. The method can be used to improve an existing structure or function of a protein. For example, a binding site for an antibody or antibody fragment or affinity for a pre-existing antigen, effector function and / or improved stability can be introduced. Alternatively, the introduction of additional "catalytically important" amino acids into a catalytic domain of an enzyme can be performed resulting in a modified or enhanced activity towards a substrate. Alternatively, completely whole structures, specificities or activities can be introduced into a polypeptide. The de novo synthesis of enzymatic activity can also be achieved. The new structures can be built on the natural or consensus "structure" of an existing protein by mutation of only relevant regions by the method of the invention. 7. Mutagenesis by review to prepare new or improved antibodies The method of this invention is used specifically to modify antibody molecules. As used in this specification, the antibody or antibody molecules refer to antibodies or portions thereof, such as full-length antibodies, Fv molecules, or other antibody fragments, individual chains or fragments thereof (e.g. , a single chain of Fv), individual chain antibodies, and chimeric antibodies. Alterations may be introduced in the variable region and / or in the conserved (constant) framework region of an antibody. Modification of the variable region can produce antibodies with better antigen binding properties, and, if desired, catalytic properties. Modification of the conserved framework region can also lead to the improvement of the cyclic-physical properties, such as solubility or stability (eg, half-life), which are especially useful, for example, in commercial production, bioavailability, effector function ( for example, complement activation and / or ADCC) and binding affinity (eg, specificity) for the antigen. Typically, mutagenesis will direct the Fv region of the antibody molecule, ie, the structure responsible for the antigen binding activity that is composed of variable regions of two chains, one of the heavy chain (VH) and one of the chain light (VL). Once the antigen binding characteristics are identified, the variable region (s) can be modified by genetic engineering in an appropriate antibody class such as IgG, IgM, IgA, IgD, or IgE. 8. Mutagenesis by Revision to Prepare / Improve Catalytic / Enzymatic Polypeptides The method of the invention is also particularly suitable for the design of catalytic proteins, particularly catalytic antibodies. Currently, catalytic antibodies can be prepared by an adaptation of conventional somatic cell fusion techniques. In this procedure, an animal is immunized with an antigen that looks like the transition state of the desired substrate to induce the production of an antibody that binds the transition state and catalyzes the reaction. The cells that produce antibodies are harvested from the animal and condensed with an immortalization cell to produce hybrid cells. The cells are then selected for the secretion of an antibody that catalyzes the reaction. This method depends on the availability of analogs of the transition state of a substrate. The procedure may be limited because such analogues are likely to be difficult to identify or synthesize in most cases. The method of the invention provides a different approach that eliminates the need for and analogous to the transition state. By the method of the invention, an antibody can be made catalytic by introducing suitable amino acids into the binding site of an immunoglobulin (Fv region). The antigen binding site (Fv) is composed of six hypervaratable loops (CDR). ), three derivatives of the immunoglobulin heavy chain (H) and three of the light chain (L), which connects beta chains with each subunit. The amino acid residues of the CDR loops contribute almost entirely to the characteristics of each specific monoclonal antibody. For example, catalytic triads (constituted by amino acid residues serine, histidine, and aspartic acid) modeled after serine proteases can be created in the hypervariable regions of the Fv region of an antibody with known affinity for the substrate molecule and selected for Proteolytic activity of the substrate. In particular, the method of the invention can be used to produce many different enzymes or catalytic antibodies, including oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Among these classes, of particular importance will be the production of proteases, carbohydrases, lipases, dioxygenases and improved peroxidases. These and other enzymes that can be prepared by the process of the invention have important commercial applications for enzyme conversions in healthcare, cosmetics, food, brewing, detergents, environment (eg, wastewater treatment), agriculture, tanning, textiles, and other chemical procedures. These include, but are not limited to, diagnostic and therapeutic applications, conversions of fats, carbohydrates and protein, degradation of organic contaminants and synthesis of chemical agents. For example, therapeutically effective proteases with fibrinolytic activity, or activity against viral structures necessary for affectivity, such as viral coat proteins, can be engineered. Such proteases may be useful anti-thrombolytic agents or anti-viral agents against viruses such as, for example, HIV, rhinovirus, influenza, or hepatitis. In the case of oxygenases (for example, dioxygenases), a class of enzymes that require a co-factor for oxidation of aromatic rings and other double bonds, industrial applications in biopulping processes, conversion of biomass into fuels or other chemical agents , conversion of wastewater pollutants, bioprocessing of coal, and detoxification of dangerous organic compounds are possible applications of novel proteins. The present invention is illustrated further in the following examples, which should not be considered as limiting.

Exemplification Throughout the examples, the following examples and procedures were used unless otherwise specified.

Materials and methods In general, the practice of the invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, PCR technology, immunology (especially, for example, antibody technology), systems of expression (eg, cell-free expression, phage display, ribosome representation, and Profusion ™), and any necessary cell cultures that are within skill in the art and are explained in the literature. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989): DNA Cloning, Volumes 1 and 2, (D. N. Giover, Ed. 1985); Oligonucleotides Synthesis (M. J. Gait, Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry,. Beaucage, Ed. John Wiley and Sons (1999) (Editor); Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford Univ. Press (1999); PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press (1990); PCR Essential Techniques: Essential Techniques, Burke, Ed., John Wiley and Sons Ltd (1996); The PCR Technique: RT - PCR, Siebert, Ed, Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies; A Laboratory Manual, Harlow et al., C. S. H. L. Press, Pub. (1999): Current Protocols in Molecular Biology, eds, Ausubel et al., John Wiley and Sons (1992); Large - Scale Mammalian Cell Culture Technology, Lubieniecki, A., Ed., Marcel Dekker, Pub., (1990). Phage Display: A Laboratory Manual, C. Barbas (Ed.), CSHL Press, (2001); Antibody Phage Display, P. O'Brien (Ed.), Humana Press (2001); Border et al., Yeast surface display for screening combinatorial polypeptide libraries, Nature Biotechnology, 15 (6): 553-7 (1997; Border et al., Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol ., 328: 430-44 (2000); representation of ribosomes as described by Pluchthum et al., In the United States Patent No. 6,348,315, and Profusion ™ as described by Szostak et al., U.S. Patent Numbers 6,258,558; 6.261, 804; and 6,214,553.

EXAMPLE 1 Mutagenesis by review of the three defined regions RN an antigen binding molecule In this example, mutagenesis is described by reviewing three CDRs of an antibody to improve the binding and proteolysis of a substrate. In particular, mutagenesis is performed "by review" of three complementarity determining regions (CDRs) of a monoclonal antibody. CDR1, CDR2, and CDR3 of the variable region of the heavy chain (VH) are defined regions selected for revision mutagenesis. For this modality, the predetermined amino acids selected are the three residues of the catalytic triad of the serine proteases, Asp, His and Ser. Asp is selected for VH CDR1, His is selected for VL CDR2, and Ser ase selects for VH CDR3. The selection of three predetermined amino acids allows us to use a convenient protease assay in order to detect when the three residues are correctly positioned to show a functional activity, i.e., proteolysis of a test substrate. An exemplary antibody, MCPC 603, is recognized as a good model for investigating binding and catalysis because the antibody binding region has been well characterized. The amino acid sequence and DNA sequence of MCPC 603 VH and VL regions are publicly available (see, for example, Rudikoff, S. and Potter, M., Biochemistry 13: 4033 (1974); Plukthun, A. et al. , Cold Spring Harbor Symp. Quant. Biol., Vol. Lll: 105-112 (1987)). The CDRs for the MCPC 603 antibody have been identified as shown in Figure 2. In the heavy chain, positions 31-35 of the amino acid residues of the CDR1 stretches, positions 50-69 of the CDR2 stretches, and the positions 101 - 111 of the CDR3 sections. In the light chain, the amino acid residues of CDR1 are 24-40, amino acids 55-62 of the CDR2 stretches, and amino acids 95-103 of the stretches of CDR3.

The design of the oligonucleotides for screening mutagenesis in the CDRs of MCPC 603 is such that the polypeptide analogs are found to have the amino acid sequence for CDR1, CDR2, and CDR3 as shown in Figures 3-5. It is understood that the synthesized oligonucleotides may be larger than the CDR regions to be altered to facilitate insertion into the target construct as shown in Figure 7. An individual chain antibody format is chosen for convenience in subsequent expression and selection steps. Oligonucleotides can be converted to double-stranded chains by enzymatic techniques (see, for example, Oliphant, AR et al., 1986, supra) and then ligated into a restricted plasmid as shown in Figure 8. Restriction sites they can be sites of natural origin or restriction sites modeled by genetic engineering. The polynucleotides encoding the polypeptide analogs can be expressed in any of convenient expression systems described herein and selected using the serine protease assay described, for example, in U.S. Patent No. 5,798,208 (see also Figures 16 - 17). Briefly, the expressed polypeptide analogs are exposed to a test substrate and examined for proteolysis of the test substrate. The amount of proteolysis, revealed by a zone of elimination of! substrate, indicates a polypeptide analog having the desired functional activity.

EXAMPLE 2 Mutagenesis by review of six defined regions in an antigen-binding molecule In this example, the mutagenesis by review of the six CDRs of an antibody is described to improve binding and proteolysis of a substrate. In particular, a mutagenesis is carried out by "revision" of the six hypervariable regions or regions determining complementarity (CDR) of the above-mentioned model antibody (MCPC 603). In this example, "revision" mutagenesis is carried out from two to three times with a different amino acid in a given region or domain. For example, Asp ,, Ser and His are sequentially in the waiting path of heavy and light chains as shown in Figure 10. Mutagenesis of continuous residues within a region may be desirable if known, or if You can deduce, that certain waste in the region will not participate in the desired function. In addition, the number of analogs can be minimized. Other considerations in the selection of the predetermined amino acid and the particular positions to be altered are that the residues may be capable of hydrogenating bonds with each other. This consideration may impose a proximity restriction on the desired variants. Thus, only certain positions within the CDRs can allow the amino acids of the catalytic triad to interact appropriately. In this way, the molecular model or other structural information can be used to enrich functional variants. In this case, the known structural information was used to identify residues in the regions that may be sufficiently close to allow the hydrogen bond between Asp, His and Ser, as well as the range of residues to be mutagenized. Roberts et al. Have identified regions of close contact between portions of the CDRs (Roberts, V. A. et al., Proc. Nati. Acad.

Sci. USA 87: 6654-6658 (1990)). This information along with the data from the x-ray structure of MCPC 603 is used to select promising areas of close contact between the CDRs targeted for mutagenesis. This type of mutagenesis by revision guided by structural / model information can be termed as "through guide" mutagenesis. Revision mutagenesis is carried out as illustrated in Figure 10 in which each CDR is subjected to a predetermined resulting amino acid that produces the 8x10E5 polypeptide analogs and if the twenty amino acids are screened, analogs of 5x10E13 polypeptides. The polynucleotides can be expressed if any of the convenient expression systems described herein and selected using the serine protease described, for example, in U.S. Patent No. 5,798,208. Briefly, the expressed polypeptide analogs are exposed to a test substrate and examined to evaluate proteolysis of the test substrate. The amount of proteolysis, revealed by a substrate removal zone, indicates a polypeptide analog having the desired functional activity.

EXAMPLE 3 Mutagenesis by review of anti-tnf binding molecules to improve function In this example, mutagenesis by review of an anti-TNF antibody to improve binding is described. In particular, mutagenesis is performed by "review" of the six hypervariable regions or complementarity determining regions (CDRs) of two anti-TNF antibodies. Anti-TNF antibodies have general application in the treatment of immune disease in patients having inappropriate levels of the TNF ligand (tumor necrosis factor). There are two commercially available anti-TNF antibodies. For convenience in carrying out mutagenesis by screening and subsequent selection, the light and heavy chain variable regions (SEQ ID Nos. 2-4) of these antibodies were converted into single chain format using a poly Gly-Ser linker ( see figure 15). The defined regions selected by screening mutagenesis with a predetermined amino acid are identified by the presence of a black bar as shown in Figure 15. These defined regions correspond to the CDRs of the individual chain antibodies. Polynucleotides representing the six CDR regions and flanking regions sufficient to allow assembly of the polypeptides into the complete individual chain sequence shown in Figure 15 are synthesized as described herein. The predetermined amino acid residues are selected for each CDR region and separately and sequentially introduced at each amino acid position through the six CDR regions. The polynucleotides are further engineered to serve as templates capable of supporting the transcription of a corresponding RNA transcript that can then be translated into a polypeptide using ribosome representation. The polynucleotides encoding the corresponding polynucleotide analogs are expressed using a transcription without cells and translation extracts. The RNA transcripts are covalently linked to a detectable moiety such as a fluorescent moiety. Alternatively, the sequence of interest can be fused in frame to a fluorescent moiety such as a green fluorescent protein (GFP) to allow convenient detection of expression and normalization of binders against non-binders. Preferably, the polynucleotides encoding the polypeptide analogs are at least partially ordered, for example, expressed in a well having multiple polypeptide analogs but can be easily depleted. In accordance with the above, each well contains a subset of polypeptide analogs now linked to the corresponding transcript attached to a fluorescent moiety using ribosome representation. The well is probed with target ligand, i.e. TNF and assayed for analogs of polypeptides that bind and with which the binding affinity compared to the wild-type polypeptide. The polypeptide analogs that bind better than the wild-type polypeptides are then further engineered into a full-length IgG antibody format for parallel assay with commercial antibody analogs for improved binding using conventional techniques.

EXAMPLE 4 Mutagenesis by review of antibodies against botulinum nerve toxin serotype B (BoNT / B) and botulinum toxin serotype A (BoNT / A) to improve function In this example, improved antibodies against botulinum nerve toxin Bb serotype (BoNT / B) and botulinum toxin serotype A (BoNT / A) are generated using revision mutagenesis (LTM) to improve function. The LTM approach is based on the creation of simple mutations by CDR through the binding pocket, based on a subset (the set of LTM) of the 20 amino acids that explore the characteristics of size, charge, hydrophobicity and hydrogen bonding. The criteria for selecting the amino acids in the LTM set are described below. 1. Purification of antibodies and gene sequencing and design of individual chains (scFv) Murine antibody fragments (Fab) with BoNT / B binding affinity can be obtained as described in Emaneul et al., (1996) Journal of Immunological Methods 193: 189-197. using BotFab 20 antibodies (SEQ ID Nos. 12 and 13, light and heavy chain polypeptides, respectively), BotFab 20 (SEQ ID Nos. 14 and 15, light and heavy chain polypeptides, respectively). Foreign sequences are described in U.S. Patent No. 5,932,449, the contents of which are incorporated herein by reference. The BoNT / B antigen (full-length toxin, light chain, and / or heavy chain) can be obtained from Metabiologics, Inc., Wl. The anti-BoNT / A antibodies described in Pless et al., (2001) Infecí. Immun. 69: 570 can be used with the aim of improving their affinities by at least an order of magnitude. The heavy chain binding domain antigen BoNT / A (BoNT / A) can be obtained from Metabiologics, Une, Wl. The V and VH fragments of the antibody (s) are cloned and sequenced using conventional molecular biology techniques. The variable regions of the molecules are amplified by the polymerase chain reaction (PCR) and ligated with a poly-Gly-Ser linker (typically SGGGGSGGGGSGGGGS (SEQ ID No. 7)) to generate single chain antibodies (csFv) . A poly-His tag (HHHHHH (SEQ ID No. 8)) and a myc tag (EQKLISEEDL (SEQ ID No. 9)) are also appended to the C-terminus of the genes to facilitate purification and detection.

These molecules are shown in any of the well-known technologies (e.g., yeast, bacteria or phage-based technologies) and were tested for their ability to bind to BoNT / B or BoNT / A. The monovalent version of scFv of whole antibodies provide a good format for undertaking mutagenesis studies and are known to generally reproduce the binding mechanism of the entire molecule, with the exception of the effects of avidity shown by multivalent molecules. 2. Antibody Enhancement Using Revision Mutagenesis (LTM) and Gene Design For Revision Mutagenesis (LTM), the following nine amino acids and their representative functional characteristics are chosen: Alanine and Leucine (aliphatic), Serine (hydroxyl group), Aspartic acid (acid) and Glutamine (amide), Lysine and Histidine (basic), Tyrosine (aromatic), Proline (hydrophobic). These amino acids show adequate chemical diversity in size, charge, hydrophobicity, and hydrogen bonding to provide significant initial information on the chemical functionality necessary to improve the antibody properties. The choices are also based on the frequency of appearance of their amino acids in the CDRs of the antibodies. For example, given between tyrosine and phenylalanine to represent amino acids with aromatic side chains, the former is chosen because of its significantly higher preponderance in the CDRs of antibodies and its capacity to bond by hydrogen. LTM is initially used to identify specific amino acids and chemical properties that are beneficial for binding, neutralization and / or any additional properties desired in the final antibody. However, LTM is not limited to these nine amino acids or nine total amino acids. The LTM analysis can be performed with any combination of amino acids and the subset of LTM can be as high as 18 amino acids (1 short of saturation mutagenesis). 2. 1 Synthesis of LTM oligonucleotides In the primary LTM analysis, the aim is to explore each contribution of the amino acid chain to the overall binding affinity within each CDR. To efficiently generate significant diversity, each of the amino acids of the LTM subset is directed at each individual position within the CDR sequence with a substitution only by CDR. In this way, each individual oligonucleotide encodes only a single CDR mutation. For example, in order to perform histidine mutagenesis of LTM over a VH CDR3 domain of hypothetical eleven amino acids, 11 oligonucleotides encoding 11 individual possible mutations are synthesized (see Figure 20). Such analysis tests the effects of having a bulky amide in each position in the CDR. Therefore, to generate a VH CDR3 LTM library, only 99 oligonucleotides are synthesized for LTM analysis (9 amino acids of LTM x 11 positions of VH CDR3). Oligonucleotide sequences are tested for accidental stop codons, wild-type duplication, inefficient codon usage, hairpins, loops, and other secondary structures using publicly available software. 2. 2 Synthesis of degenerate oligonucleotides for combinations of beneficial LTM mutations By employing the systematic LTM replacement of individual amino acids within a CDR, the preference of the chemical functionalities in each position is uncovered. In order to combine all the mutations of the LTM selections and explore the possible additiveness and energetic synergy between them, the degenerate oligonucleotides encoding these mutations and the wild-type sequence are synthesized. The degenerate combination of oligonucleotides with 1.6 x 104 variants is subsequently used to generate a second generation library that explores the additive nature of these substitutions. 2. 3 Computer-assisted oligonucleotide design, library, and database of results. The software coupled with DNA synthesizers of automatic custom constructions allows the rapid synthesis of oligonucleotides. The first stage involves the decision of which target amino acids will be incorporated into the CDRs. The software determines the codon preference (eg, yeast, bacterial, or phage codon preference, depending on the chosen system) necessary to introduce the target amino acids and also eliminates any duplication of the wild-type sequence that can be generated by this design procedure. It is then analyzed to evaluate the structures of potential stop codons, hairpins, and other loops or for problematic sequences that are subsequently corrected before synthesis. The complete LTM design plan is then sent to the DNA synthesizer, which performs an automatic synthesis of the oligonucleotides. In this way, the oligonucleotides needed to create the libraries can be generated quickly. An electronic database can store information of all LTM oligonucleotide sequences, details of the CDR substitutions of the scFv libraries, and results of binding assays for a target antigen. The archiving of the oligonucleotide data allows the rational iterations of the design strategies, and the reuse of the oligonucleotides of reagents. 3. Global LTM Strategy LTM is used initially to identify specific amino acids and chemical properties that are beneficial for binding properties, neutralization and / or any desired additional properties in the final antibody. It also quickly identifies regions that do not tolerate any mutagenesis without significant loss of affinity (or any physical property that is selected). Therefore, not only is the LTM analysis a good methodology to explore the chemical requirements in each position in all the CDRs of an antibody, but it also rapidly determines the amino acids absolutely required for antibody binding. After identification of the beneficial mutations by LTM, the combinatorial mutagenesis schemes can be used to first incorporate all of these different amino acid mutations to generate multiply mutated CDRs. Additionally or alternatively, standby mutagenesis (WTM) can be used to probe the effects of multiple mutations of the same amino acid in a CDR (as described, for example, in U.S. Patent Nos. 5,830,650, 5,798,208). 4. LTM scFv libraries The LTM techniques described above are used to create combinations of oligonucleotides with mutations in a single CDR of the light or heavy chain. These oligonucleotides are synthesized to include some of the surrounding conserved frames to facilitate superposition and hybridization during PCR. These combinations of oligonucleotides are used to generate all possible VL and VH chains in which there are mutations in single, double, and triple CDRs (single, double, and triple combinations of CDR1, 2, and 3) using single superposition extension PCR (SOE-PCR) (as described in Horton et al., (1989) Gene 77: 61-68). SOE - PCR is a fast and simple procedure to combine DNA fragments that do not require restriction sites, restriction endonucleases, or DNA ligases. In two SOE-PCR regions in the gene they are first amplified by PCR using primers designed so that the PCR products share a complementary sequence at one end. Under these PCR conditions, the complementary sequences hybridize, forming an overlap. The complementary sequences then act as primers, allowing extension by DNA polymerase to produce a recombinant molecule. For example, to create the combination of VH chains in which both CDR-H1 and CDR-H2 are mutated and CDR-H3 is wild-type, which is called "110" (1 means a mutant CDR and oO means one Wild-type CDR), the CDR-H1 mutant genes are used as templates and SOE-PCR is carried out to bind the CDR-H2 oligonucleotides to generate the doubly mutated combination (Figure 21). Considering that each CDR can be either wild type or mutant, there are seven possible combinations (shown by arrows in Figure 21) for each of the combinations of the V and V chains (not including the wild type molecule "000" ). The combination of the seven combinations V and VH creates 63 VL-VH combinations of non-wild type (scFv), figure 21). Each of the 64 VL and VH combinations (including the wild-type sequence) is referred to as a "subset" of the entire LTM scFv library set. A set of the scFv library is created for each amino acid selected for substitution. The number of amino acid sequences represented within each library subset depends on the length of the CDR, the amino acid sequence within the CDR, and the oligonucleotide design strategy of LTM.

. Selection of libraries for review and selection of improved antibodies A variety of procedures are available for the expression and representation of antibodies. These include bacteriophage, Escherichia coli, and yeast. Although each of these methods has been used for antibody enhancement, the yeast representation system produces several advantages (Power and Wittrup (1997) Nat. Biotechnol 15: 553-557). The yeast can easily accommodate library sizes of up to 107, with 103 - 105 copies of each antibody displayed on each cell surface. Yeast cells can be easily selected and separated using flow cytometry and separation of fluorescence activated cells (FACS) or magnetic beads. Yeasts also produce rapid selection and re-growth. The eukaryotic secretion system and yeast glycosylation pathways allow a much larger subset of scFv molecules to fold correctly and show on the cell surface than prokaryotic display systems. Yeast representation coupled to direct evolution has been used to increase the KD of an scFv antibody fragment to fluorescein at 48 fM, two orders of magnitude stronger than any monovalent ligand previously reported (Border et al., (2000) PNAS 97: 10701-10705). The representation system uses the agglutinin a yeast adhesion receptor to represent proteins on the cell surface. The proteins of interest, in the present case, libraries of anti-BoNT / B scFv LTM or libraries of anti-BoNT / A scFv LTM, are expressed as fusion partners with the Aga2 protein. These fusion proteins are selected from the cell and become disulfide-bound to the Aga1 protein, which binds to the wall of the yeast cells (see Invitrogen, bibliography of the yeast representation product pYD1). In addition, there are included carboxyl terminal labels that can be used to control expression levels and / or normalize binding affinity measurements. Magnetic beads coated with streptavidin (Spherotech) are used to screen and select antibodies that bind to BoNT / B or BoNT / A with high affinity. This methodology employs binding of high affinity antibodies to biotinylated antigen which is then bound to streptavidin-coated beads in order to select the yeast clones (Yeung and Wittrup (2002) Biotechnol. Prog 18: 212-220) and Feldhaus et al. ., (2003) Nature Biotech.1V. 163-170). The BoNT / A or BoNT / A polypeptide (Metabiologics) is biotinylated using conventional protocols (Pierce) and the screening is performed using procedures well known in the art.

Using selection based on balance and kinetics, antibodies with improved affinities are selected from these libraries. The effectiveness of each round of selection is controlled by analytical FACS (FACScan). In addition, the relative binding affinities of the individual molecules represented on the yeast surface are measured by antigen titration. This allows rapid identification of molecules with improved affinity. The scFv clones are then sequenced to identify beneficial mutations. 6. Generation of soluble antibodies for BIAcore affinity measurements Antibodies of interest are subcloned in soluble expression systems (Picchia pastoris and / or E. coli) and soluble protein is generated. Commercially available vectors and cell lines for expression of soluble antibodies exist, including those of Invitrogen (e.g., pPlC9 for P. pastoris) and Novagen (pET20b for periplasmic expression in E. coli). These systems are routinely used to generate soluble individual or full-length chain antibodies. The expression system of P. pastoris (Invitrogen) routinely produces 1-5 mg per liter of soluble purified scFv. The purification of proteins is facilitated by the presence of His tag at the C-terminus of the molecule, in the case of individual chains or by protein A or protein G columns for full-length antibodies. Single chain or soluble full length antibodies are generated to obtain BIAcore affinity kinetic rate measurements. This stage is necessary as high affinity scFv molecules on the cell surface of yeast clones should be verified as a soluble molecule without cells. The soluble single-chain and full-length antibodies generated in the above manner can be used to obtain BIAcore affinity measurements, as well as in the neurite outgrowth assay or the mouse lethality assay described below. 7. Selection of selected antibodies for neutralization in vivo or in vitro A cell-based assay that uses the outward growth of neurites of chicken prime neurons as an indicator of BoNT intoxication and, thus, as a means of quantifying toxin neutralization it can be used to select the selected antibodies. Preliminary experiments using cultures of dorsal root ganglia explants of fertilized chicken eggs have indicated that there was less axonal outgrowth of plants treated with BoNT / A than controls. Alternatively, a chicken ciliary gaglio-iris muscle neuromuscular joint assay (as described in Lomneth et al., (1990) Neuroscience Letters 113: 211-216) can be used. The mouse lethality assay (MLA, as described, for example, Schantz and Kautter (1978) J. Assoc. Off.Anal.Chem. 61: 96-99) is another well-known and accepted in vivo method for testing neutralization of BoNT. The assay involves the interperitoneal injection of approximately 0.5 ml of sample preparations of BoNT / B or BoNT / A with and without the antibody in 20 to 30 grams, white mice of the ICR strain. Mortality due to respiratory failure is observed for 1 - 4 days. The quantification of neutralization requires serial dilutions of the MAbs with varying levels of LD 5o of BoNT / B or mouse BoNT / A. The MLA is possessed to be used to determine the neutralization capacity of Iso optimized antibodies identified in the in vitro selection. The ability to neutralize antibodies can also be measured in vitro by the mouse protection assay (MPA, Goeschel et al., (1997) Exp. Neurol. 147: 96-102). In the MPA, the left phrenic nerve, together with the left hemidiagramam, is excised from the mouse. The phrenic nerve is electrostimulated continuously in a tissue bath. The purified antibodies are incubated with BoNT / B or BoNT / A and added to the tissue bath. Toxin-induced paralysis is defined as a 50% reduction in the initial muscle tremor.

Equivalents Those skilled in the art will recognize, or be able to ascertain using nothing more than routine experimentation, many equivalents to the specific embodiments of the invention, described in this specification. Such equivalents are intended to be encompassed by the following claims. Having described the invention as above, the content of the following claims is declared as property.

Claims (61)

NOVELTY OF THE INVENTION CLAIMS

1. A method of generating a library of analogs of polypeptides in which a predetermined amino acid appears at each position in a defined region of the polypeptide comprising: selecting a defined region of the amino acid sequence of the polypeptide; determining an amino acid residue to be substituted at each amino acid position within the defined region; synthesizing individual polynucleotides encoding the defined region, the polynucleotides representing collectively possible variant polynucleotides according to the following criteria: i) containing each polynucleotide at each codon position in the defined region, or a codon required for the amino acid residue of the polypeptide or a codon for the predetermined amino acid residue, and ii) contain each polynucleotide no more than one codon for the predetermined amino acid residue thereby generating a polynucleotide library in which the predetermined amino acid residue appears at each amino acid position within the defined region.

2. The method according to claim 1, further characterized in that the polynucleotides are combined together.

3. The method according to claim 1, further characterized in that two or more regions defined within the polypeptide are mutagenized.

4. - The method according to claim 31, further characterized in that the same predetermined amino acid is selected for substitution within each of the two or more defined regions.

5. The method according to claim 3, further characterized in that different predetermined amino acids are selected for substitution within each of the two or more defined regions, respectively.

6. The method according to any one of the preceding claims, further characterized in that the defined region or defined regions comprises a functional domain of the polypeptide.

7. The method according to claim 6, further characterized in that the functional domain is selected from the group consisting of an antibody binding site, a conserved antibody framework region, an antibody effector region, a binding site of receptor, and a catalytic site.

8. The method according to claim 7, further characterized in that the antibody binding site or portion thereof comprises a CDR domain selected from the group consisting of CDR1, CDR2, CDR3, CDR4, CDR5, CDR6 and a combination of the same.

9. The method according to claim 7, further characterized in that the conserved framework region of antibody comprises a domain selected from the group consisting of FR1, FR2, FR3, FR4 and a combination thereof.

10. The method according to claim 7, further characterized in that the antibody effector region comprises a domain selected from the group consisting of a complement binding site and an Fc binding region.

11- The method according to claim 1, further characterized in that the predetermined amino acid residue is selected from the group consisting of Ser, Thr, Asn, Gln, Cys, His, Asp, Lys, Arg, Ala, Gly, Lie, Leu , Met, Phe, Pro, Trp, and Val.

12. The method according to any one of the preceding claims, further characterized in that the defined region comprises at least 3 to 40 amino acids.

13. The method according to any one of the preceding claims, further characterized in that the polynucleotides are synthesized as an expression library.

14. The method according to any one of the preceding claims, further characterized in that the polynucleotides are synthesized using enzymatic means.

15. The method according to any one of the preceding claims, further characterized in that the polynucleotides are synthesized using polymerase chain reaction.

16. The method according to any one of the preceding claims, further characterized in that the expression library is selected from the group consisting of a phage display library, a ribosome / polysome representation library, a library for representation of yeasts, a bacteria representation library and an organized representation library.

17. A library of polypeptide analogs by the method described in claim 1.

18. A method of identifying a polypeptide having a desired structure or function comprising: selecting a defined region of the amino acid sequence of the polypeptide. polypeptide; determining an amino acid residue to be substituted at each amino acid position within the defined region; synthesizing individual polynucleotides encoding the defined region, the polynucleotides representing collectively possible variant polynucleotides according to the following criteria: i) containing each polynucleotide at each codon position in the defined region, or a codon required for the amino acid residue of the polypeptide or a codon for one of the predetermined amino acid residue, and ii) containing each polynucleotide no more than one codon for the predetermined amino acid residue, thereby generating a polynucleotide library containing the polynucleotides; expressing the expression library to produce polypeptide analogs; and selecting the polypeptide analogs to select a polypeptide having a desired structure or function.

19. The method according to claim 18, further characterized in that the method further comprises the step of identifying the polynucleotide encoding the selected polypeptide analogue.

20. The method according to claim 18, further characterized in that the selection comprises, contacting a polypeptide with a target substrate, the polypeptide associated with the polinucieotide encoding the polypeptide, the polynucleotide additionally comprising a detectable moiety, so that a variant polypeptide capable of binding to a target substrate is detected and therefore is identified as encoded by the polynucleotide.

21. The method according to claim 20, further characterized in that the detectable moiety is selected from the group consisting of a fluorescent moiety, a UV moiety, and a moiety that absorbs visible light.

22. The method according to claim 20, further characterized in that the detectable moiety is selected from the group consisting of a biotin moiety, a GST moiety, and a His tag moiety.

23. The method according to claim 20, further characterized in that the polynucleotide is associated with the polypeptide analogue using ribosome representation.

24. The method according to claim 18, further characterized in that two or more regions defined within the polypeptide are mutagenized.

25. - The method according to claim 24, further characterized in that the same predetermined amino acid is selected for substitution within each of the two or more defined regions.

26. The method according to claim 24, further characterized in that the different predetermined amino acids are selected for substitutions within each of the os or more defined regions.

27. The method according to claim 18, further characterized in that the polypeptide is a single-chain antibody (sFVs).

The method according to claim 18, further characterized in that the defined region comprises a functional domain of the polypeptide.

29. The method according to claim 18, further characterized in that the defined region comprises a CDR or potion thereof selected from the group consisting of CDR1, CDR2, CDR3, CDR4, CDR5, CDR6 and a combination thereof.

30. The method according to claim 18, further characterized in that the defined region is a conserved framework region of antibody comprising a domain selected from the group consisting of FR1, FR2, FR3, FR4 and a combination thereof.

31. The method according to claim 18, further characterized in that the antibody effector region comprises a domain selected from the group consisting of a complement binding site and an Fc binding region.

32. The method according to claim 18, further characterized in that the predetermined amino acid residue is selected from the group consisting of Ser, Thr, Asn, Gln, Cys, His, Asp, Lys, Arg, Ala, Gly, lie, Leu, Met, Phe, Pro, Trp, and Val.

33. A library of polynucleotides encoding polypeptide analogs comprising one or more defined regions in which a predetermined amino acid residue is substituted at each amino acid position within the defined region, the polynucleotides collectively representing all possible variants according to the following criteria: i) each polynucleotide contains at each codon position in the defined region, either a codon required for the amino acid residue of the polypeptide or a codon for one of the predetermined amino acid residue, and ii) each polynucleotide contains no more than one codon for the predetermined amino acid residue.

34. The library according to claim 33, further characterized in that two or more regions defined within the polypeptide are mutagenized.

35. The library according to claim 33, further characterized in that the same predetermined amino acid is selected for substitution within each of the two defined regions.

36. The library according to claim 33, further characterized in that the different predetermined amino acids are selected for substitution within each of the two defined regions.

37.- The library according to claim 33, further characterized in that the library is an expression library.

38.- The library according to claim 33, further characterized in that the expression library is selected from the group consisting of a phage display library, a ribosome / polysome representation library, a yeast representation library, a bacteria representation library and an organized representation library.

39. The library according to claim 33, further characterized in that the polynucleotides additionally comprise one or more transcriptional regulatory elements.

40. The library according to claim 39, further characterized in that the polynucleotides, when transcribed and translated in vitro, are associated with the polypeptides encoded by the corresponding polynucleotides.

41. The library according to claim 40, further characterized in that the polynucleotides are associated with the polypeptide using ribosome / polysome representation.

42. The library according to claim 41, further characterized in that the polynucleotides comprise RNA.

43. - The library according to claim 42, further characterized in that the polynucleotides additionally comprise a detectable moiety.

44. The library according to claim 43, further characterized in that the detectable moiety comprises a fluorescent moiety.

45. The library according to claim 33, further characterized in that the library comprises at least 106 different polynucleotides.

46. The library according to claim 33, further characterized in that the library comprises at least 45-1012 different polynucleotides.

47. The library according to claim 33, further characterized in that the polypeptide encodes a binding polypeptide.

48. The library according to claim 47, further characterized in that the binding polypeptide is selected from the group consisting of a heavy chain variable region (VH), a light chain variable region (VL), and an antibody of a single chain (sFv).

49. The library according to claim 33, further characterized in that the polypeptide encodes an enzyme.

50.- The library according to claim 33, further characterized in that the polypeptide encodes an enzyme inhibitor.

51. The library according to claim 33, further characterized in that the polypeptide encodes a catalytic polypeptide.

52. The library according to claim 33, further characterized in that the library is immobilized on a solid support.

53. The library according to claim 33, further characterized in that the solid support is a microchip.

54.- The library according to claim 33, further characterized in that the library is an ordered library.

55.- A microchip comprising a series of immobilized polynucleotides according to the library defined in claim 33.

56.- A polypeptide analogue identified according to the library defined in claim 33, wherein the polypeptide binds to a target molecule and comprises a binding region selected from the group consisting of a heavy chain variable region (VH), a light chain variable region (VL), and a single chain antibody (sFv).

57. The polypeptide analogue according to claim 56, further characterized in that the specified target molecule is TNFa.

58. The polypeptide analogue according to claim 56, further characterized in that the polypeptide has at least 70% identity with the amino acid decency of SEQ ID No. 1, 2, 3, 4, 5 or 6.

59.- A method of generating a subset of polypeptide analogs having a desired structure or function comprising: selecting a defined region of the amino acid sequence of the polypeptide; determining an amino acid residue to be substituted at each amino acid position within the defined region; synthesizing individual polynucleotides encoding the defined region, the polynucleotides representing collectively possible variant polynucleotides according to the following criteria: i) containing each polynucleotide at each codon position in the defined region, or a codon required for the synthesis of the amino acid residue of the polypeptide or a codon for the predetermined amino acid residue, and i) contain each polynucleotide no more than one codon for the predetermined amino acid residue thereby generating an expression library containing the polynucleotide; exposing the expression library to conditions under which the library is expressed; selecting the library expressed to identify a polypeptide having a desired structure or function; comparing the structure or function of the polypeptide when compared to a control criterion, in which a polypeptide that corresponds or exceeds the control criterion is classified as an effector and a polypeptide that fails the control criterion is classified as a non-effector; categorize effectors and non-effectors in a database; and consult the database to determine the sequence of a subset of polypeptides to be synthesized.

60.- The method according to claim 59, further characterized in that one or more of the previous steps is computerized.

61. The method according to claim 59, further characterized in that the control criterion is selected from the group consisting of binding affinity, stability and effector function. 62.- The method according to claim 59, further characterized in that the criterion is catalytic activity on a specified substrate. 63.- The method according to claim 59, further characterized in that the polypeptide is selected from the group consisting of a heavy chain variable region (VH), a light chain variable region (V), and a single-chain antibody. chain (sFv). 64.- A means suitable for use in an electronic device that has instructions for carrying out one or more steps of the procedure described in claim 59.