(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`(19) World Intellectual Property Organization
`International Bureau
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`(43) International Publication Date
`2 March 2006 (02.03.2006)
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`(51)
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`(21)
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`International Patent Classification:
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`CIZP 21/06 (2006.01)
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`C12N 15/74 (2006.01)
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`(81)
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`International Application Number:
`PCT/US2005/024140
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`(22)
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`International Filing Date:
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`6 July 2005 (06.07.2005)
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`(25)
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`Filing Language:
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`(26)
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`Publication Language:
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`(30)
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`Priority Data:
`60/585,918
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`English
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`English
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`(34)
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`6 July 2004 (06.07.2004)
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`US
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`(71)
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`(72)
`(75)
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`Applicant #or all designated States except US): BIOREN
`INC. [US/US]; 100 Glenn Way, Suite #1, San Carlos, CA
`94070—6264 (US).
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`Inventor; and
`(for US only): CREA, Roberto
`Inventor/Applicant
`[IT/US]; 700 Occidental Avenue, San Mateo, CA 94402
`(US).
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`(10) International Publication Number
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`WO 2006/023144 A2
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`Designated States (unless otherwise indicated, for ever
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ,
`OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, U7., VC,
`VN, YU, ZA, ZM, ZW.
`
`Designated States (unless otherwise indicated, for ever
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
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`Published:
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`without international search report and to be republished
`upon receipt of that report
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`(74)
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`Agents: REMILLARD, Jane, E. et al.; Lahive & Cock—
`field, LLP, 28 State Street, Boston, MA 02109 (US).
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`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin—
`ning ofeach regular issue ofthe PCT Gazette.
`
`(54) Title: LOOKJl‘HROUGH MUTAGENESIS FOR DEVELOPING AL’l‘ERED POLYPEPl‘lDES WITH ENHANCED PROPr
`ERTIES
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`(57) 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 is disclosed. The method is based on the premise that certain amino acids play a crucial role in the structure and function
`of proteins and thus is capable of identifying and distinguishing functional amino acid residues ("hot spots") from non—functional
`amino acids residues ("cold spots") within a polypeptide or portion thereof. 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.
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`LOOK-THROUGH MUTAGENESIS FOR DEVELOPING ALTERED
`POLYPEPTIDES WITH ENHANCED PROPERTIES
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`Related Information
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`The entire contents of all other patents, patent applications, and references cited
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`throughout the following specification also are incorporated by reference herein in their
`entireties.
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`Backgmund of the Invention
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`Mutagenesis is a powerful tool in the study of protein structure and function.
`Mutations can be made in the nucleotide sequence of a cloned gene encoding a protein
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`of interest and the modified gene can be expressed to produce mutants of the protein.
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`By comparing the properties of a wild-type protein and the mutants generated, it is often
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`possible to identify individual amino acids or domains of amino acids that are essential
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`for the structural integrity and/or biochemical function of the protein, such as its binding
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`and/or catalytic activity. The number of mutants that can be generated from a single
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`protein, however, renders it difficult to select mutants that will be informative or have a
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`desired property, even if the selected mutants that encompass the mutations are solely in
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`putatively important regions of a protein (e. g, regions that make up an active site of a
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`protein). For example, the substitution, deletion, or insertion of a particular amino acid
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`may have a local or global effect on the protein.
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`Previous methods for mutagenizing polypeptides have been either too restrictive,
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`too inclusive, or limited to knocking out protein function rather than to gaining or
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`improving function. For example, a highly restrictive approach is selective or site-
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`'directed mutagenesis which is used to identify the presence of a particular functional site
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`or understand the consequences of making a very specified alteration within the
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`functional site. A common application of site directed mutagenesis is in the study of
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`phosphoproteins where an amino acid residue, that would ordinarily be phosphorylated
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`and allow the polypeptide to carry out its function, is altered to confirm the link between
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`phosphorylation and functional activity. This approach is very specific for the
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`polypeptide and residue being studied.
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`Conversely, a highly inclusive approach is saturation or random mutagenesis that
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`is designed to produce a large number of mutations encompassing all possible alterations
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`within a defined region of a gene or protein. This is based on the principle that, by
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`generating essentially all possible variants of a relevant protein domain, the proper
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`arrangement of amino acids is likely to be produced as one of the randomly generated
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`mutants. However, in practice, the vast number of random combinations of mutations
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`generated can prevent the capacity to meaningfully select a desired candidate because of
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`the presence of the so-called “noise” of so many undesired candidates.
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`Another approach, referred to as “Walk Through” mutagenesis (see, e. g. , US.
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`Patent Nos: 5,830,650; 5,798,208) has been used to mutagenize a defined region of a
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`polypeptide by synthesizing a mixture of degenerate oligonucleotides that, statistically,
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`contain a desired set of mutations. However, because degenerate polynucleotide
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`synthesis is employed, Walk—Through mutagenesis yields a number of undesired
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`alterations in addition to the desired set of mutations. For example, to sequentially
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`introduce a mutation across a defined region of only five amino acid positions, a set of
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`over 100 polynucleotide must be made (and screened) (see, e.g., Fig. 6). Accordingly,
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`to make and screen, for example, two or three regions becomes increasingly complex,
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`i. e. , requiring the making and screening of 200 to over 300 polynucleotides,
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`respectively, for the presence of only 10 to 15 mutations.
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`In yet another approach which has been used to mutagenize proteins is alanine
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`seaming mutagenesis, where an alanine residue is “scanned” through a portion of a
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`protein to identify positions where the protein’s function is interrupted. However, this
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`approach only looks at loss of protein function by way of substituting a neutral alanine
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`residue at a given position, rather than gain or improvement of function. Thus, it is not a
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`useful approach for generating proteins having improved structure and function.
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`Accordingly, a need remains for a systematic way to mutagenize a protein for
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`new or improved function.
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`Summary of the Invention
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`The invention pertains to a method of mutagenesis for the generation of novel or
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`improved proteins (or polypeptides) and to libraries of polypeptide analogs and specific
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`polypeptides generated by the methods. The polypeptide targeted for mutagenesis can
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`be a natural, synthetic or engineered polypeptide, including fragments, analogs and
`mutant forms thereof.
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`In one embodiment, the method comprises introducing a predetermined amino
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`acid into essentially every position within a defined region (or several different regions)
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`of the amino acid sequence of a polypeptide. A polypeptide library is generated
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`containing polypeptide analogs which individually have no more than one
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`predetermined amino acid, but which collectively have the predetermined amino acid in
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`every position within the defined region(s). Alone, this method can be referred to as
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`“look-through” mutagenesis because, in effect, a single, predetermined amino acid (and
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`only the predetermined amino acid) is substituted position-by—position throughout one or
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`more defined region(s) of a polypeptide.
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`However, in a preferred embodiment, the LTM method is improved by using it to
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`identify functional amino acids (or so-called “hot spots”) from non-functional amino
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`acids (or so—called “cold Spots”) within a polypeptide, or portion thereof, to further
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`reduce the number of residues to be altered in order to screen and obtain a desired
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`property in a polypeptide. Accordingly, the improved method of look—through
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`mutagenesis (LTM) (hereafter the improved LTM being referred to as LTM2) allows for
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`the identification and building of a subset of candidate molecules representing only the
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`most relevant functional alterations in the polypeptide which can then be efficiently
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`screened free of any “noise”. Importantly, LTM2 also allows for the construction of an
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`LTM2 library having superior advantages over traditional libraries because it has been
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`designed to include only alterations in the amino acid residues of the polypeptide most
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`likely to have an effect on the function of the polypeptide and therefore, upon screening,
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`more likely to yield an altered polypeptide having an enhanced property. Thus, LTM2
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`allows one to “look-through” the structural and functional consequences of separately
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`substituting a predetermined amino acid at each functional amino acid position within a
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`defined region of the polypeptide, thereby segregating a specific protein chemistry to the
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`defined region without any interference or “noise” from the generation of unwanted
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`polypeptide analogs (i.e., analogs containing amino acid substitutions other than those
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`that follow the LTM2 scheme) (see, for example, Fig. 1).
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`Accordingly, the present invention allows for highly efficient and accurate
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`systematic evaluation of the role of a specific amino acid change in one or more defined
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`regions of a polypeptide. This becomes particularly important when evaluating (by
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`mutating) two or more defined regions, such that the number of polypeptide analogs
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`required greatly increases and, thus, the presence of undesired analogs also increases.
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`The present invention obviates this problem by completely eliminating undesired
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`analogs and, thus, the potential that any changes in protein structure or function
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`observed are the result of anything but substitution of the predetermined amino acid.
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`Thus, the effect of segregating a specific protein chemistry to even multiple regions with
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`a protein can be studied with high accuracy and efficiency. Importantly, this includes
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`. studying how mutagenesis can effect the interaction of such regions, thereby improving
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`the overall structure and function of the protein.
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`In a particular embedment of the invention, the methods of the invention are
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`suitable for identifying a particular chemical motif that maps to one or more functional
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`amino acid resides or positions. The amino acid residue(s) that contribute to such a
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`chemical motif can occur at one or more positions that are contiguous, non-contiguous,
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`within one or more CDR regions, and/or within one or more polypeptides, for example,
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`antibody heavy and light chains. The methods of the invention allow for the further
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`exploration of a chemical motif in that they allow for the systematic testing (or chemical
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`profiling) of related amino acid chemistries at selected amino acid position(s) or defined
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`region(s). Accordingly, in one embodiment, the invention provides a method for
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`identifying a desired chemistry and then exploring the consequences of incorporating
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`related or unrelated chemistries to achieve either an enhanced property or to remove a
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`deleterious property. Typical amino acid side chain chemistries suitable for profiling by
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`the methods of the invention are polar, positively charged, negatively charged, and
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`hydrophobic amino acid side chain chemistries. In one embodiment, a charged
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`chemistry is identified as resident at a selected amino acid reside(s), position, or defined
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`region(s) and other charged amino acids are substituted for the parental amino acid such
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`that an alteration in a measurable property is achieved.
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`In a preferred embodiment, the
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`alteration in a measurable property is an enhanced property in an antibody, for example,
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`improved antigen-binding or effector function.
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`Accordingly, the invention also provides antibody libraries comprising related
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`amino acid side group chemistries introduced at selected amino acid positions(s) /
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`defined regions having, for example, related chemistry, for the efficient screening of
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`antibodies with improved properties.
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`In another embodiment of the invention, the library of polypeptide analogs is
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`generated and screened by first synthesizing individual polynucleotides encoding a
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`defined region or regions of a polypeptide where, collectively, the polynucleotides
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`represent all possible variant polynucleotides according to the look-through criteria
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`described herein. The method is used to identify and distinguish functional amino acid
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`residue(s) (positions) from non—functional amino acid residue(s) (positions). A subset of
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`variant polynucleotides are expressed, for example, using in vitro transcription and
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`translation and/or using a display technology, such as ribosome display, phage display,
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`bacterial display, yeast display, arrayed display, or any other suitable display system
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`known in the art.
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`The expressed polypeptides are then screened and selected using functional
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`assays, such as binding assays or enzymatic/catalytic assays. In one embodiment, the
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`polypeptides are expressed in association with the polynucleotide that encodes the
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`polypeptide, thereby allowing for identification of the polynucleotide sequence that
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`encodes the polypeptide.
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`In yet another embodiment, the polypeptides are directly
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`synthesized using protein chemistry.
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`In yet another embodiment of the invention, a combinatorial beneficial library of
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`the V1, and V” CDR amino-acid sequence variations is constructed. This second library
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`is constructed by generating coding sequences having, at each amino acid variation
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`position, codons for the wildtype amino acid and for each of the previously identified
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`beneficial variant amino acids at that position.
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`Thus, the present invention provides a method of intelligent mutagenesis that can
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`be used to generate libraries of polypeptide analogs that are of a practical size for
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`screening, in part, because the libraries are devoid of any undesired analog polypeptides
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`or so-called noise. The method can be used to study the role of specific amino acids in
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`polypeptide structure and function and to develop new or improved polypeptides such as
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`antibodies, binding fragments or analogs thereof, single chain antibodies, catalytic
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`antibodies, enzymes, and ligands. In addition, the method can be performed with the
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`benefit of a priori information, e. g., via computer modeling, that can be used to select an
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`initial subset of polypeptide analogs to be produced and studied using LTM2.
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`Other advantages and aspects of the present invention will be readily apparent
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`from the following description and Examples.
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`Brief Description of the Figures
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`Figure 1 illustrates the advantages of improved LTM (LTM2) over LTM in that
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`functional amino acids are distinguished from non-functional amino acids such that a
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`more beneficial subset of candidate molecules is obtained and screened.
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`Figure 2 illustrates a general approach for the use of polymerase chain reaction
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`(PCR) to build defined regions of an antibody heavy and light chain for identifying
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`functional amino acid residues, into a larger gene context.
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`Figure 3 illustrates the arrangement of variable light-chain (V1,) and variable
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`heavy chain (VH) CDRs in a synthetic single chain antibody (scFv) anti-ovalbumin gene
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`context.
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`In the application of LTM, a leucine amino acid is introduced into each of the
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`fourteen residues 56-69 in VH CDR2 of the antibody. For. the application of LTM2, only
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`those residues identified as functional are further explored by mutagenesis.
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`Figure 4 illustrates the by single overlap extension polymerase chain reaction
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`(SOE-PCR) for the production of an LTM VH CDR2 library; the production of multiple
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`LTM VH CDR libraries; and an array of LTM library combinations containing both VH and
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`VL CDRs
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`Figure 5 illustrates the diversity of the libraries of the invention with the x and y
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`axes of the matrix representing the CDRs of each of the light and heavy chains wherein an
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`“0” indicates a wild-type CDR and a “l ” indicates a mutated CDR and the intersected
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`number representing the complexity of the resultant subset library (e. g, 4 means four
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`CDRs are simultaneously mutated).
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`Figure 6 shows a schematic of a yeast expression vector for displaying proteins of
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`interest, e. g., polypeptide analogs of the invention, on the surface of yeast for efficient
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`identification of function (phenotype) and corresponding encoding sequence (genotype).
`Figure 7 represents a Fluorescence-Activated Cell Sorter (FACSTM) plot of the
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`binding of biotinylated ovalbumin and streptavidin FITC to wild type anti—ovalbumin
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`scFv (gray line); pYDl vector alone (solid gray area); and control scFv (black line).
`Figure 8 represents Fluorescence—Activated Cell Sorter (FACSTM) plots showing
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`a selection gate (the R1 trapezoid) for identifying only those LTM clones that expressed
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`the scFv fusion with a higher binding affinity to ovalbumin than the anti—ovalbumin wild
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`type antibody (left panel), the distribution of binding affinities of the total LTM library
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`(center panel), and a post sort FACS analysis (right panel) to confirm that >80% of the
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`pre-screen anti-ovalbumin scFv clones were within the predetermined criteria.
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`Figure 9 illustrates steps in the screening of scFv antibodies (e. g, anti—ovalbumin)
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`formed in accordance with the present invention for improved binding affinity based on
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`equilibrium binding kinetics (e. g., to ovalbumin).
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`Figure 10 shows equilibrium binding curves for anti—ovalbumin scFv expressing
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`cells prior to selection (circles), after one round of selection (light triangles), after two
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`rounds of selection (dark triangles), and for the anti-ovalbumin wild—type reference
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`antibody (black squares).
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`Figure 11 illustrates typical steps for screening of antibodies formed in accordance
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`with the present invention for high binding affinity based on particular binding kinetics,
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`e.g., antibody'K0fl~constants, using the test antigen ovalbumin.
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`Figure 12 shows the identification of enhanced properties in two clones (Le,
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`higher relative Km as compared to an reference antibody (square)) using the methods of
`the invention.
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`Figure 13 represents the enhanced properties (see fold better than wild type) of a
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`subset of improved clones having lower EC50 values with respect to an anti—ovalbumin
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`wild-type reference antibody control (square).
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`Figure 14 shows a matrix representing the functional (hot spots) and non-functional
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`(cold spots) amino acid positions of an exemplary antibody. Mutations associated with
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`enhanced affinity (relative to the reference wild type antibody) based on equilibrium
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`binding (ECso) and/0r kinetic binding experiments are shown below each VH and VL CDR
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`position.
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`Detailed Description of the Invention
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`In order to provide a clear understanding of the specification and claims, the
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`following definitions are provided below.
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`Definitions
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`As used herein the term “analog” refers to a variant or mutant polypeptide (or a
`nucleic acid encoding such a polypeptide) having one or more amino acid substitutions.
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`The term “binding molecule” refers to any binding molecule, including proteins,
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`polypeptides, and peptides that bind to a substrate or target.
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`In one embodiment, the
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`binding molecule is an antibody or binding fragment thereof (e. g. , a Fab fragment),
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`single domain antibody, single chain antibody (e. g., scFv), or peptide capable of binding
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`a ligand.
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`In another embodiment, the binding molecule, in particular, binding molecules
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`comprising CDR region(s), can comprise nontraditional scaffolds or framework regions
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`derived from other antibodies, immunoglobulins, or immunoglobulin-like molecules
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`(e. g., fibronectin), or be in part or in whole, of synthetic origin.
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`The term “defined region” refers to a selected region of a polypeptide.
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`Typically, the defined region includes all or a portion of a functional site, e. g., the
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`binding site of a ligand, the binding site of a binding molecule or receptor, or a catalytic
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`site. The defined region may also include multiple portions of a functional site. For
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`example, the defined region can include all, a portion, or multiple portions of a
`complementarity determining region (CDR), e. g., a single domain binding region, or a
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`complete heavy and/or light chain variable region (Fv) of an antibody. Thus, a
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`functional site may include a single or multiple defined regions that contribute to the
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`functional activity of the molecule.
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`The terms “functional amino acid(s)” and “non-functional amino acid(s)” refer
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`to, respectively, the amino acid residues (or corresponding amino acid residue position)
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`within a polypeptide (or portion thereof) that are determined (using, for example, the
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`methods of the invention) to contribute to a measurable property or activity of the
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`polypeptide. Accordingly, a functional amino acid residue(s) (or corresponding
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`position(s)) is referred to as a “hot sp0t(s)” as it is a residue or residue position that
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`influences the activity of the polypeptide as compared to a non—functional residue(s) or
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`position(s) which does not influence the activity of the polypeptide and therefore
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`referred to as a “cold spot(s)”. A functional amino acid residue (or position) is
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`distinguished from a non-functional amino acid residue (or position) as being suitable
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`for mutagenesis. Typically, when applying the methods of the invention to the
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`investigation of an antibody molecule, amino acid residues that alter, for example,
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`antigen binding, are considered functional residues/positions (i. 6., hot spots) whereas
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`residues that do not alter such binding are referred to as non—functional
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`residues/positions (i. e. , cold spots).
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`The term “measurable property” refers to a functional property or activity of a
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`polypeptide (or portion thereof) that can be measured, determined, or assayed for, using
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`standard techniques and include, binding activity, kinase activity, catalytic activity,
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`thermal stability, or enzymatic activity. Measurable properties of polypeptides that are
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`antigen-binding polypeptides, e.g., antibodies, typically include binding specificity,
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`binding avidity, binding affinity, Fc receptor binding, glycosylation, complement
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`binding, half-life stability, solubility, thermal stability, catalytic activity, and enzymatic
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`activity.
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`- The term “look-through mutagenesis” or “LTM” refers to a method for
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`introducing a predetermined amino acid into essentially every position within a defined
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`region (or several different regions) of the amino acid sequence of a polypeptide. A
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`polypeptide library is generated containing polypeptide analogs which individually have
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`no more than one predetermined amino acid, but which collectively have the
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`predetermined amino acid in every position within the defined region(s).
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`The term “improved look-through mutagenesis” or “LTM2” refers to LTM
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`conducted so as to identify or distinguish functional amino acid residues (hot spots) from
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`non-functional amino acid residues (cold spots). Accordingly, the LTM2 method allows
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`for selectively introducing a predetermined amino acid into the functional amino acid
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`residue positions within a polypeptide (or portion thereof). Corresponding LTM2
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`libraries are therefore enriched for polypeptides analogs having amino acid alterations
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`most likely to confer an altered or enhanced property. LTM2 can be carried out
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`subsequent to LTM or based on a priori information as to the functionality of a given
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`amino acid residue or residue position.
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`The term “library” refers to two or more molecules mutagenized according to the
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`method of the invention. The molecules of the library can be in the form of
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`polynucleotides, polypeptides, polynucleotides and polypeptides, polynucleotides and
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`polypeptides in a cell free extract, or as polynucleotides and/or polypeptides in the
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`context ofa phage, prokaryotic cells, or in eukaryotic cells; Libraries of the invention
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`can contain 2 or more molecules or polypeptide analogs, for example about 2 to 10,
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`about 10 to 50, about so to 102, about 103, about 10“, about 105, about 106, about 107,
`about 108, about 109, about 1010, about 10”, about 1012 , about 10”, or more, or any
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`interval or range of the foregoing.
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`The term “mutagenizing” refers to the alteration of an amino acid sequence.
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`This can be achieved by altering or producing a nucleic acid (polynucleotide) capable of
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`encoding the altered amino acid sequence, or by the direct synthesis of an altcrcd
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`polypeptide using protein chemistry.
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`The term “mutagenesis” refers to, unless otherwise specified, any art recognized
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`technique for altering a polynucleotide or polypeptide sequence. Preferred types of
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`mutagenesis include walk-through mutagenesis (WTM), beneficial walk-through
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`mutagenesis, look-through mutagenesis (LTM), improved look-through mutagenesis
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`(LTM2), or combinations thereof.
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`The term “combinatorial beneficial mutagenesis” refers to a combination library
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`of coding sequences that encode degenerate mixtures of VL and/or V“ CDR amino-acid
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`sequence variations initially identified from the predetermined LTM amino acid
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`mutagenesis screen as having an alteration on a measurable property.
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`In the
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`combinatorial beneficial mutation approach, oligonucleotide coding sequences are
`generated which represent combinations of these beneficial mutations identified by LTM.
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`These combinations may be combinations of different beneficial mutations within a single
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`CDR, mutations within two or more CDRs within a single antibody chain, or mutations
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`within the CDRS of different antibody chains.
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`PCT/US2005/024140
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`The term “polynucleotide(s)” refers to nucleic acids such as DNA molecules and
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`RNA molecules and analogs thereof (e. g. , DNA or RNA generated using nucleotide
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`analogs or using nucleic acid chemistry). As desired, the polynucleotides may be made
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`synthetically, e. g. , using art—recognized nucleic acid chemistry or enzymatically using,
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`e. g., a polymerase. Typical modifications include methylation, biotinylation, and other
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`art-known modifications.
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`In addition, the nucleic acid molecule can be single-stranded
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`or double-stranded and, where desired, linked or associated (e. g., covalently or non-
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`covalently) to a detectable moiety.
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`The term “variant polynucleotide” refers to a polynucleotide encoding a
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`corresponding polypeptide analog (or portion thereof) of the invention. Thus, variant
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`polynucleotides contain one or more codons that have been changed to result in
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`expression of a different amino acid.
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`The term “polypeptide(s)” refers to two or more amino acids joined by a peptide
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`bond, 8. g. , peptides (e. g, from 2 to ~50 amino acid residues), as well as longer peptide
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`sequences e. g., protein sequences which typically comprises amino acid sequences from
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`as few as 50 amino acid residues to more than 1,000 amino acid residues,
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`The term “pooling” refers to the combining of polynucleotide variants or
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`polypeptide analogs to form libraries representing the look-through mutagenesis (LTM)
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`or improved look—though mutagenesis (LTMZ) of an entire polypeptide region. The
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`molecules may be in the form of a polynucleotide and/or polypeptide and may coexist in
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`the form of a sublibrary, as molecules on a solid support, as molecules in solution,
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`and/or as molecules in one or more organisms (e.g., phage, prokaryotic cells, or
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`eukaryotic cells).
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`The term “predetermined amino acid” refers to an amino acid residue selected
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`for substitution at each position within a defined region of a polypeptide to be
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`mutagenized. This does not include position(s) within the region that already (e.g.,
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`naturally) contain the predetermined amino acid and, thus, which need not be substituted
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`with the predetermined amino acid. Accordingly, each polypeptide analog generated in
`accordance with the present invention contains no more that one “predetermined amino
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`acid” residue in a given defined region. However, collectively, the library of
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`polypeptide analogs generated contains the predetermined amino acid at each position
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`within the region being mutagenized, and in a preferred embodiment, at amino acid
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`positions determined to be functional (hot spots). Typically, a predetermined amino
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`acid is selected for a particular size or chemistry usually associated with the side group
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`of the amino acid. Suitable predetermined amino acids include, for example, glycine
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`and alanine (sterically small); serine, threonine, and cysteine (nucleophilic); valine,
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`leucine, isoleucine, methionine, and proline (hydrophobic); phenylalanine, tyrosine, and
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`tryptophan (aromatic); aspartate and glutamate (acidic); asparagine, glutamine, and
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`histidine (amide); and lysine and arginine (basic). Use of non-traditional amino acid
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`residues (e. g. , homocysteine) are also within the scope of the invention and can be
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`introduced using any art recognized techniques.
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`Detailed Description
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`The study of proteins has revealed that certain amino acids play a crucial role in
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`their structure and function. For example, it appears that only a discrete number of
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`amino acids participate in the binding of an antibody to an antigen or are involved in the
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`catalytic event of an enzyme.
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`Though it is clear that certain amino acids are critical to the activity or function
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`of proteins, it is difficult to identify which amino acids are involved, how they are
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`involved, and what substitutions can improve the protein’s structure or function.
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`In part,
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`this is due to the complexity of the spatial configuration of amino acid side chains in
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`polypeptides and the interrelationship of different portions of the polypeptide that
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`contribute to form a functional site. For example, the interrelationship between the six
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`CDRs of the variable heavy and light chain regions of an antibody contribute to the
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`antigen or ligand-binding pocket.
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`Previous mutagenesis methods, such as selective (site-directed) mutagenesis and
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`saturation mutagenesis, are of limited utility for the study of protein structure and
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`function in view of the enormous number of possible variations in complex
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`polypeptides. This is especially true given that desirable combinations are often
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`accompanied by the presence of vast amounts of undesirable combinations or so-called
`noise.
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`The method of this invention provides a systematic, practical, and highly
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`accurate approach for evaluating the role of particular amino acids and their position,
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`within a defined region of a polypeptide, in the structure or function of the polypeptide
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`and, thus, for producing improved polypeptides.
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`1. Selecting a Defined Region
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`In accordance with the present invention, a defined region or regions within a
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`protein are selected for mutagenesis. Typically, the regions are believed to be important
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`to the protein’s structure or funct