AU2012204022C1

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Publication number: AU2012204022C1
Application number: AU2012204022A
Authority: AU
Inventors: Sara C. Birtalan; Frederic A. Fellouse; Sachdev S. Sidhu
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2005-12-02
Filing date: 2012-07-06
Publication date: 2014-02-20
Anticipated expiration: 2026-12-01
Also published as: AU2012204022A1; AU2012204022B2

Abstract

The invention provides antibodies or antigen binding fragments thereof to DR5 and HER-2. The antibodies and/ or antigen binding fragments thereof comprise variant CDRs comprising highly restricted amino acid sequence diversity. The invention also provides 5 these polypeptides as fusion polypeptides to heterologous polypeptides such as at least a portion of phage or viral coat proteins, tags and linkers. In addition, compositions and methods of use for treatment of cancer and immune related conditions are provided. 350000_1 (GHMatters) P77907.AU.1

Description

do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of 5 the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof. In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and 10 heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB- strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995). The present invention provides an expression system in which the quantitative ratio of 15 expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components. One technique for modulating translational strength is disclosed in Simmons et al., 20 U.S. Pat. No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which 25 can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgarno sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a "codon bank" at the beginning of a coding sequence that does not change the amino acid sequence of the signal 30 sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the 128 3500906_1 (GHMatters) P77907.AU1 bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS. A Companion to Methods in Enzymo 1. 4:151-158. In certain embodiments, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited sel provides a comparison of expression levels of each 5 chain as well as the yield of the desired antibody products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5, 840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention. 10 Prokaryotic host cells suitable for expressing antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one 15 embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3 110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3 110 AjhuA (AtonA) ptr3 lac Iq lacL8 20 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 3;1,446), E. coli B, E. colix 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known. in the art and described in, for example, Bass et al., 25 Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and 30 additional protease inhibitors may desirably be incorporated in the cell culture. 129 3500900_1 (GHMatters) P77907.AU.1 Antibody Production Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. 5 Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs 10 polyethylene glycol/DMSO. Yet another technique used is electroporation. Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of 15 the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene. Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture 20 with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol. The prokaryotic host cells are cultured at suitable temperatures. For E coli growth, for example, the temperature ranges from about 20*C to about 39*C, from about 25 0 C to 25 about 37 0 C, and/or about 30 0 C may be used. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. For E. coli, the pH can be about 6.8 to about 7.4, and can be about 7.0. If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. In one 30 aspect of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. The phosphate-limiting medium can be C.R.A.P medium (see, e.g., 130 3500906_1 (GHMatlers) P77907.AU.1 Simmons et al., J. Immunol. Method-, (2002), 263:133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art. In one embodiment, the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves 5 disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant can be 10 filtered and concentrated for further purification of the produced proteins. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay. In one aspect of the invention, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available 15 for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity; in certain embodiments, the large-scale fermentors have about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (a common carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in 20 volumetric capacity, and can range from about I liter to about 100 liters. In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD 5 50 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and 25 described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction times may be used. To improve the production yield and quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors 30 overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host 131 3500908_1 (GHMatters) P77907.AU.1 cells. Chen et al. (1999) JBio Chem 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramrn and Pluckthun (2000) J. Biol. Chem. 275:17106 17113; Arie et al. (2001) Mol. Microbiol. 39:199-210. 5 To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some 10 E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the 15 expression system of the invention. Antibody Purification In one embodiment, the antibody protein produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are 20 exemplary of suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75. In one aspect, Protein A immobilized on a solid phase is used for immunoaffinity 25 purification of the antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. In certain embodiments, the solid phase to which Protein A is immobilized is a column comprising a glass or silica surface. In certain embodiments, the solid phase to which Protein A is immobilized is a controlled pore glass 30 column or a silicic acid column. In some applications, the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants. As the first step of purification, the preparation derived from the cell culture as described above is applied onto the Protein A immobilized solid phase to allow specific 132 3500906_1 (GHMatters) P77907.AU.1 binding of the antibody of interest to Protein A. The solid phase is then washed to remove contaminants non-specifically bound. to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution. 5 Generating antibodies using eukaryotic host cells: The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. (i) Signal sequence component 10 A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. In certain embodiments, the heterologous signal sequence selected is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, 15 for example, the herpes simplex gD signal, are available. The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody. (ii) Origin of replication Generally, an origin of replication component is not needed for mammalian 20 expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter. (iii) Selection gene component Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or 25 other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media. One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein 30 conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin. Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as 133 3500906_1 (GHMatters) P77907.AU.1 DHFR, thymidine kinase, metallothionein-I and -II (e.g., primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a 5 competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096). Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR 10 protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199. (iv) Promoter component 15 Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody polypeptide nucleic acid. Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many 20 genes is a CNCAAT (SEQ ID NO:585) region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO:586) sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors. Antibody polypeptide transcription from vectors in mammalian host cells is 25 controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host 30 cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIlI E 134 350001 (GHMatter) P77007.AU.1 restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human p-interferon cDNA in mouse cells under the control of a 5 thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. (v) Enhancer element component Transcription of DNA encoding the antibody polypeptide of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many 10 enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 15 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-encoding sequence. In certain embodiments, the enhancer is located at a site 5' from the promoter. (vi) Transcription termination component Expression vectors used in eukaryotic host cells will typically also contain sequences 20 necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone 25 polyadenylation region. See W094/11026 and the expression vector disclosed therein. (vii) Selection and transformation of host cells Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of 30 useful mammalian host cell lines are monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. 135 350090_1 (GHMatters) P77907.AU.1 Nall. Acad Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, 5 ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N. Y. Acad Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Host cells are transformed with the above-described expression or cloning vectors for 10 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. (viii) Culturing the host cells The host cells used to produce an antibody of this invention may be cultured in a 15 variety of media. Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 20 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as 25 GENTAMYCNTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be 30 apparent to the ordinarily skilled artisan. ix) Purification of antibody When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, 136 350090_1 (GHMatters) P77907AU.1 the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore 5 Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. 10 The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human y1, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is 15 attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX

TM

resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion 20 exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. Following any preliminary purification step(s), the mixture comprising the antibody 25 of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5. In certain embodiments, the low pH hydrophobic interaction chromatography is performed at low salt concentrations (e.g., from about 0-0.25M salt). Activity Assays 30 The antibodies of the present invention can be characterized for their physical/chemical properties and biological functions by various assays known in the art. The purified immunoglobulins can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing 137 3500090_1 (GHMatters) P77907.AU.1 size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion. In certain embodiments, the immunoglobulins produced herein are analyzed for their biological activity. In some embodiments, the immunoglobulins of the present invention are 5 tested for their antigen binding activity. The antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immuntoassays. 10 The antibodies or antigen binding fragments can be further selected for functional activity, for example, antagonist or agonist activity. For example, anti-HER-2 antibodies can be selected for the ability to inhibit tyrosine phosphorylation of HER-2, inhibit proliferation of cancer cells or to induce apoptosis of cancer cells. Assays for identifying and measuring these activities are described for example in W098/17797 . 15 In addition, anti-DR5 antibodies can be selected for the ability to induce apoptosis of cancer cells and/ or inhibit the function of inflammatory cells. In other embodiments, anti DR5 antibodies are selected for the ability to compete with Apo-2L for binding to DR5. In yet other embodiments, anti-human DR5 antibodies are selected for binding to marine and/ or cynomolgous DR5. Assays for determining biological activity can be conducted using 20 methods known in the art, such as DNA fragmentation (see, e.g., Marsters et al., Curr. Biology, 6: 1669 (1996)), caspase inactivation, DR5 binding (see, e. g., WO 98/51793, published Nov. 19, 1998. Apoptosis can be measured by identifying condensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be determined and 25 measured, for instance, by cell viability assays (such as Alamar blue assays or MTT assays), FACS analysis, caspase activation, DNA fragmentation (see, for example, Nicoletti et al., J. Immunol. Methods, -139:271-279 (1991), and poly-ADP ribose polymerase, "PARP", cleavage assays known in the art. In one embodiment, as assay for apoptosis involves making two fold serial dilutions 30 of control standard and an anti-DR5 antibody in 96-well tissue culture plates . 5 Apo-2 ligand (amino acids 114-281, described in PCT USOO/17579) is tested for comparison. Colo-205 (20000 cells-well) human colon carcinoma cells (ATCC) are seeded into the 96-well plates. The plates are incubated at 37 for 24 hours. AlamazBlue (Trek Diagnostic Systems, Inc.) is 138 3500906_1 (GHMatters) P77907.AU.1 added to the wells for the last 3 hours of the 24 hours incubation time. Fluorescence is read using a 96-well fluorometer with excitation at 530 nm and emission of 590 nm. The results are expressed in relative fluorescence units (RFU). In one embodiment, the present invention contemplates an altered antibody that 5 possesses some but not all effector functions, which make it a desired candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In certain embodiments, the Fc activities of the produced immunoglobulin are measured to ensure that only the desired properties are maintained. In vitro and/or in vivo cytotoxicity assays can be 10 conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is summarized in Table 3 15 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in US Patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model 20 such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). CIq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. To assess complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding and in vivo clearance/half life determinations can also be performed using methods 25 known in the art, e.g., those described in the Examples section. Humanized Antibodies The present invention encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non 30 human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et a. (1988) Nature 332:323-327; Verhoeyen et al. (1988) 139 3500G06_1 (GHMatters) P77907.AU.1 Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non 5 human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called 10 "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:90 1). Another method uses a particular framework derived from the consensus 15 sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623). It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one 20 method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected 25 candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the 30 target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. 140 350001 (GHMaters) P77907.AU.I Antibody Variants In one aspect, the invention provides antibody fragments comprising modifications in the interface of Fc polypeptides comprising the Fc region, wherein the modifications facilitate and/or promote heterodimerization. These modifications comprise introduction of a 5 protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance is positionable in the cavity so as to promote complexing of the first and second Fc polypeptides. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168. In some embodiments, amino acid sequence modification(s) of the antibodies 10 described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences 15 of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made. To increase the half-life of the antibodies or polypeptide containing the amino acid 20 sequences of this invention, one can attach a salvage receptor binding epitope to the antibody (especially an antibody fragment), as described, e.g., in US Patent 5,739,277. For example, a nucleic acid molecule encoding the salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence of this invention so that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding 25 epitope and a polypeptide sequence of this invention. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fe region of an IgG molecule (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie, V et al., (2000) Ann. Rev. Immunol. 18:739-766, Table 1). Antibodies with substitutions in an Fc region thereof and increased serum half-lives are also described in 30 WOOO/42072 (Presta, L.), WO 02/060919; Shields, R.L., et al., (200 1) JBC 276(9):6591 6604; Hinton, P.R., (2004) JBC 279(:8):6213-6216). In another embodiment, the serum half life can also be increased, for example, by attaching other polypeptide sequences. For example, antibodies of this invention or other polypeptide containing the amino acid 141 350006.1 (GHMattr) P77907.AU.1 sequences of this invention can be attached to serum albumin or a portion of serum albumin that binds to the FcRn receptor or a serum albumin binding peptide so that serum albumin binds to the antibody or polypeptide, e.g., such polypeptide sequences are disclosed in WOO1/45746. In one embodiment, the serum albumin peptide to be attached comprises an 5 amino acid sequence of DICLPRWGCLW (SEQ ID NO:608). In another embodiment, the half-life of a Fab according to this invention is increased by these methods. See also, Dennis, M.S., et al., (2002) JBC 277(38):35035-35043 for serum albumin binding peptide sequences. A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described 10 by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other 15 variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity. 20 Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include 25 the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but 30 FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in the table 142 35009061 (GHMatters) P77907.AU.1 below, or as further described below in reference to amino acid classes, may be introduced and the products screened. Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; GIn; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; GIn; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine 5 Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a 143 35009061 (GHMatters) P77907.AU.1 sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73 75, Worth Publishers, New York (1975)): 5 (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q) (3) acidic: Asp (D), Glu (E) (4) basic: Lys (K), Arg (R), His(H) Alternatively, naturally occurring residues may be divided into groups based on 10 common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; 15 (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites. 20 One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. 25 Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites 30 for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring 144 350090_1 (GHMatters) P77907.AU.1 residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. 5 Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant 10 version of the antibody. It may be desirable to introduce one or more amino acid modifications in an Fc region of the immunoglobulin polypeptides of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGI, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a 15 substitution) at one or more amino acid positions including that of a hinge cysteine. In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody used in methods of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, for example in the Fc region. These antibodies would nonetheless retain substantially the same characteristics 20 required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) Cl q binding and/or Complement Dependent Cytotoxicity (CDC), for example, as described in W099/51642. See also Duncan & Winter Nature 322:738-40 (1988); US Patent No. 5.,648,260; US Patent No. 5,624,821; and W094/29351 25 concerning other examples of Fc region variants. Immunoconjugates The invention also pertains to immunoconjugates, or antibody-drug conjugates (ADC), comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of 30 bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer (Syrigos and 145 3500906_1 (GHMatters) P77007.AU.I Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. patent 4,975,278) theoretically allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of 5 toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby. Both polyclonal antibodies and monoclonal antibodies have been 10 reported as useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 15 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA 20 binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. ZEVALIN@ (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope conjugate composed of a murine IgC 1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and mIn or 90Y 25 radioisotope bound by a thiourea linker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL), administration results in severe and prolonged cytopenias in most patients. MYLOTARGTM (gemtuzumab 30 ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; US Patent Nos. 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab 146 35009061 (GHMatters) P77907.AU.1 mertansine (Immunogen, Inc.), an antibody drug conjugate composed of the huC242 antibody linked via the disulfide linker SPP to the maytansinoid drug moiety, DM1, is advancing into Phase II trials for the treatment of cancers that express CanAg, such as colon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen 5 Inc.), an antibody drug conjugate composed of the anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid drug moiety, DM1, is under development for the potential treatment of prostate tumors. The auristatin peptides, auristatin E (AE) and monomethylauristatin (MMAE), synthetic analogs of dolastatin, were conjugated to chimeric monoclonal antibodies c3R96 (specific to Lewis Y on carcinomas) and cAC 10 10 (specific to CD30 on hematological malignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784) and are under therapeutic development. Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include without limitation diphtheria A chain, nonbinding active fragments of diphtheria 15 toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of 20 radioconjugated antibodies. Examples include 2 12 Bi, 1311, 131 In, 90 Y, and 186 Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds 25 (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid 30 (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. 147 350090.1 (GHMatters) P77907.AU.I Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothecene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein. Maytansine and maytansinoids 5 In one embodiment, an antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce 10 maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the 15 disclosures of which are hereby expressly incorporated by reference. Maytansinoid-antibody conjugates In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates 20 containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 BI, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The 25 conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127 131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA. I that binds the HER-2/neu oncogene. 30 The cytotoxicity of the TA. 1 -maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 105 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansinoid drug, which 148 3500906_1 (GHMatters) P77907.AU.1 could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice. Antibody-maytansinoid conjugates (immunoconjugates) Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to 5 a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of 10 naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. In certain embodiments, maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol 15 molecule, such as various maytansinol esters. There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992). The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase 20 labile groups, or esterase labile groups, as disclosed in the above-identified patents. Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters 25 (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p diazoniumbenzoyl)-ethylenediaminei, diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In certain embodiments, coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate 30 (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [19781) and N-succinimidyl-4-(2 pyridylthio)pentanoate (SPP) to provide for a disulfide linkage. The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction 149 350090_1 (GHMatters) P77907AU.1 with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In one embodiment, the linkage is formed at the C-3 position of maytansinol or a 5 maytansinol analogue. Calicheamicin Another immunoconjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation 10 of conjugates of the calicheamicin family, see U.S. patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, yl , 2 , aL3 , N-acetyl-yl, PSAG and O' (Hinman et al., Cancer Research 53:3336 3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. 15 patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects. 20 Other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. patents 5,053,394, 5,770,710, as well as esperamicins (U.S. patent 5,877,296). 25 Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, 30 gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993. 150 3500908_1 (GHMatters) P77907.AU.1 The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuc lease; DNase). For selective destruction of the tumor, the antibody may comprise a highly 5 radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I"',2, Y 90 , Re', Re, Sm 5 3 , Bi 2 12 P , Pb 2 12 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scinti graphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, 10 MRI), such as iodine-123 again, iodine-131, indium-I 11, fluorine-19, carbon-13, nitrogen 15, oxygen-17, gadolinium, manganese or iron. The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of 15 hydrogen. Labels such as tc99m or 1123, .Re 18, Rel18 and In"' can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) describes other methods in detail. 20 Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds 25 (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p diazoniumbenzoyl)-ethylenediamine:i, diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon- 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid 30 (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, 151 3500906_1 (GHMatters) P77907 AU.1 photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020) may be used. The compounds of the invention expressly contemplate, but are not limited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, 5 MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo KMUS, sulfo-MBS, sulfo-SIAB, sullo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl (4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog. 10 Preparation of antibody drug conjugates In the antibody drug conjugates (ADC) of the invention, an antibody (Ab) is conjugated to one or more drug moieties (D), e.g. about I to about 20 drug moieties per antibody, through a linker (L). The ADC of Formula I may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the 15 art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form Ab-L, via a covaleni bond, followed by reaction with a drug moiety D; and (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form D-L, via a covalent bond, followed by reaction with the nucleophilic group of an antibody. Ab-(L-D), I 20 Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active 25 esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible inierchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive 30 thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. 152 3500906_1 (GHMatters) P77907 AU.1 Antibody drug conjugates of the invention may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which 5 may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with 10 appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852). Such aldehyde can be reacted with a drug moiety or linker nucleophile. 15 Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; 20 (iii) aldehydes, ketones, carboxyl, and maleimide groups. Alternatively, a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired 25 properties of the conjugate. In yet another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated 30 to a cytotoxic agent (e.g., a radionucleotide). 153 3500906_1 (GHMatlers) P77907AU.1 Antibody Derivatives The antibodies of the present invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. In certain embodiments, the moieties suitable for derivatization of the antibody are water soluble 5 polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3 dioxolane, poly- 1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl 10 pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may 15 vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. 20 Pharmaceutical Formulations Therapeutic formulations comprising an antibody of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions, lyophilized or 25 other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or 30 benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic 154 3500906_1 (GHMatters) P77907.AU.1 polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal 5 complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN

4 . Absorbance was determined spectrophotometrically at 450 nm. Weak cross-reactivity was defined as a signal between 0.2-2.0 and strong cross-reactivity was 10 defined as a signal about 2.0. The results for HER2 binding clones are shown in Figure 21B . As shown in Figure 25, of the SXH3 clones isolated, the S:R clones displayed the greatest average non-specific binding (0.5-0.6 OD at 450 nm by ELISA assay), while the S:W, S:Y, and S:F clones each displayed similar low levels of average non-specific binding (0-0.1 OD at 450 nm by ELISA assay). 15 A competitive phage ELISA was used to estimate the binding affinities of HER2 binding phage-displayed Fabs. Phage were produced in a 96-well format as described, and phage supernatants were serially diluted in PBT buffer, then incubated on plates coated with HER2 for 15 minutes. The plates were washed with PBS including 0.05% Tween 20 and were incubated for 30 minutes with horseradish peroxidase/anti-M 13 antibody conjugate 20 (diluted 1:5000 in PT buffer) (Pharmacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories) and quenched with 1.0 M H 3

PO

4 . Absorbance was measured spectrophotometrically at 450 nm to determine the phage concentration giving about 50% of the signal at saturation. A fixed, sub saturating concentration of phage was diluted two fold in PBT buffer or PBT buffer 25 containing two-fold serial dilutions of HER2 protein from 250 nM HER2 to 0.12 nM HER2. The mixtures were incubated for one hour with gentle shaking at room temperature, transferred to plates coated with HER2 and the plates were incubated for 15 minutes. The plates were washed and treated exactly as above. The binding affinities were estimated as

IC

PO

4 . Absorbance was determined spectrophotometrically at 450 nm. 174 3500906. (GHMatters) P77907.AU.1 Weak cross-reactivity was defined as a signal between 0.2-2.0 and strong cross-reactivity was defined as a signal above 2.0. The results for the SX-surface clones are shown in Figure 24B. As shown in Figure 27, of the SX-surface clones isolated, the S:R and S:W clones displayed the greatest average non-specific binding (0.5-0.6 OD and approximately 4.0 OD, 5 respectively, at 450 nm by ELISA assay), while the S:Y and S:F clones each displayed similar low levels of average non-specific binding (0-0.1 OD at 450 nm by ELISA assay). A competitive phage ELISA was also used to estimate the binding affinities of HER2 binding phage-displayed Fabs. Phage were produced in a 96-well format as described above, and phage supernatants were serially diluted in PBT buffer, then incubated on plates coated 10 with HER2 for 15 minutes. The plates were washed with PBS including 0.05% Tween 20 and were incubated for 30 minutes with horseradish peroxidase/anti-M13 antibody conjugate (diluted 1:5000 in PT buffer) (Phannacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories) and quenched with 1.0 M H 3

PO

4 . Absorbance was measured spectrophotometrically at 450 nm to 15 determine the phage concentration giving -50% of the signal at saturation. A fixed, sub saturating concentration of phage was diluted two fold in PBT buffer or PBT buffer containing two-fold serial dilutions of HER2 protein from 250 nM HER2 to 0.12 nM HER2. The mixtures were incubated for one hour with gentle shaking at room temperature, transferred to plates coated with HER2 and the plates were incubated for 15 minutes. The 20 plates were washed and treated exactly as above. The binding affinities were estimated as

IC

50 values (defined as the concentration of antigen that blocked 50% of the phage binding to the immobilized antigen). The results are shown in Figure 24B. Based on this analysis, the analysis of HER2-binding clones from the SXH3 library (Example 6), and the YSGR-A-D library (Example 1), soluble Fab proteins from three clones 25 (clone nos. 42 (YSGR-A) and B11 (SXH3) and G54 (SX-surface)) were purified and subjected to surface plasmon resonance analysis of binding to human HER2. BlAcore@ data was obtained according to Chen et al., J. Mol. Biol. (1999), 293(4): 865-81. Briefly, binding affinities of the purified Fabs for human HER2 were calculated from association and dissociation rate constants measured using a BIAcore@-A 100 surface plasmon resonance 30 system (BIACORE, Inc., Piscataway, N.J.). HER2 was covalently coupled to a biosensor chip at two different concentrations using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's (BIAcore, Inc., Piscataway, N.J.) Instructions. HER2 was buffer-exchanged into 10 mM 175 3500908_1 (GHMaters) P77907.AU.1 sodium acetate, pH 5.0 and diluted to approximately 2.5 or 5.0 ptg/ml. Aliquots of HER2 were injected at a flow rate of 5 p/min to achieve approximately 50-170 response units (RU) of coupled protein. A solution of 1 M ethanolamine was injected as a blocking agent. For kinetics measurements, twofold serial dilutions of each Fab were injected in HBT at 25 5 *C at a flow rate of 10 pL/minute over each flow cell. The k,, and krff values were determined from the binding curves using the BlAevaluation software package (BIACORE, Inc., Piscataway, N.J.) using two-spot global fitting and combining the data from both flow cells. The equilibrium dissociation constant, KD, was calculated as Koftrkon. The BIAcore@ data is summarized in Figure 26A and B. Clone BI I had a ka of 1.9 x 106 M's', a kd of 1.7 10 x 10- s~1, and a KD of 890 pM. Rmaxl for the clone Bl 1 experiments was 19 RU, and Rmax2 for the clone Bl 1 experiments was 29 RU. (Figure 26A) Clone G54 had a ka of 2.0 x 10 5 M-'s-', a kd of 2.2 x 10 3 s 1 , and a KD of 11 nM. Rmaxi for the clone G54 experiments was 21 RU and Rm.a for the clone G54 experiments was 34 RU. (Figure 26A) Clone YSGR-A 42 had a ka of 2.7 x 106 M~'s-', a kd of 1.5 x 10- s~', and a KD of 570 pM. Rmax l for the 15 clone 42 experiments was 25 RU, and Rmax2 for the clone 42 experiments was 38 RU. (Figure 26B)The tryptophan-containing clone (B 11) had a faster ko, and correspondingly smaller KD than the tyrosine-containing clone (G54). To study binding of anti-HER2 antibodies to HER2 expressed on mammalian cells, the binding of purified Fab protein of clones 42 (YSGR-A), B II (SXH3), G54 (SX-surface), 20 and G37 (SX-surface) to NR6 fibroblast cells over-expressing HER2 (NR6-HER2) was studied by flow cytometry. One million NR6-HER2 cells were incubated with 10 pg/ml Fab for 1 hour, followed by incubation with an Alexa488-conjugated murine anti-human IgG antibody for 1 hour. As a negative control, Fab binding to non-expressing NR6 cells was studied. As a positive control, 4D5 Fab was used. As demonstrated in Figure 27, clones 42, 25 BI 1, G54, and G37 bind specifically to Her2 on NR6 cells. A competitive ELISA was used to test binding competition with Herceptin and Omnitarg and between several HER2 clones in IgG format (see Figure 28 for the CDR sequences of the relevant clones). Biotinylated HER2 protein was serially diluted from 200 nM to 0.39 nM in PBT buffer, then incubated on plates coated with purified IgG proteins for 30 15 minutes. The plates were washed with PBS containing 0.05% Tween 20, and were incubated for 30 minutes with horseradish peroxidase/anti-M 13 antibody conjugate (diluted 1:5000 in PT buffer) (Pharmacia). The plates were washed, developed with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories) and quenched 176 3500906_1 (GHMatters) P77907 AU.1 with 1.0 M H 3

PO

4 . Absorbance was measured spectrophotometrically at 450 nm to determine the biotinylated HER2 concentration giving around 50% of the signal at saturation. A fixed, sub-saturating concentration of biotinylated HER2 was diluted two-fold in PBT buffer or PBT buffer containing 100 nM purified IgG proteins. The mixtures were incubated 5 for one hour with gentle shaking at room temperature, transferred to plates coated with IgG proteins, and the plates were incubated for 15 minutes. The plates were washed and treated as above. As shown in Figure 29, none of the HER2-binding IgGs blocked binding of biotinylated HER2 to either Omnitarg or Herceptin. The IgGs did block binding between each other in two groups. One group made up of clones Bi 1, G37, G54, and YSGR-A-42 10 compete for the same epitope and blocked binding to biotinylated HER2 that had been previously incubated with any of those clones. A second group made up of clones YSGR-A 27, B27, G43, and YSGR-D-104 compete for the same epitope on HER2 and blocked binding to biotinylated HER2. Group one clones are all higher-affinity binders than the group two clones. 15 All publications (including patents and patent applications) cited herein are hereby incorporated in their entirety by reference. TABLE 1 20 Human DRS-ECD polypeptide MSALLILALVGAAVADYKDDDDKLSALITQQDLAPQQRVAPQQKRSSPSEGLCPPG HHIS 25 EDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTVCQCEEGTFR EED SPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESGTKHSGEAPAVEETVTSSPGTP ASP CSLS (SEQ ID NO:595) 30 Human DRS Polypeptide meqrgqnapa asgarkrhgp gpreargarp glrvpktlvl vvaavlllvs aesalitqqd lapqqraapq qkrsspsegl 35 cppghhised grdcisckyg qdysthwndl lfclrctrcd sgevelspct ttrntvcqce egtfreedsp emcrkcrtgc prgmvkvgdc tpwsdiecvh kesgiiigvt vaavvlivav fvcksllwkk vlpylkgics ggggdpervd rssqrpgaed nvlneivsil qptqvpeqem evqepaeptg vnmlspgese hllepaeaer sqrrrllvpa negdptetlr qcfddfadlv pfdsweplmr klglmdneik vakaeaaghr dtlytmlikw vnktgrdasv htlldaletl gerlakqkie dhllssgkfm ylegnadsal s (SEQ ID NO:604) 40 177 3500906.1 (GHMatters) P77907.AU.1 MURINE DR5 ECD GLQRPEESPSRGPCLAGQYLSEGNCKPCREGIDYTSHSNHSLDSCILCTVCKEDKVVE 5 TR CNITTNTVCRCKPGTFEDKDSPEICQSCSNCTDGEEELTSCTPRENRKCVSKTAWAS WHK (SEQ ID NO:605) 10 Apo-2L polypeptide sequence I MetAlaMetMetGluValGlnGlyGlyProSerLeuGlyGlnThrCysValLeuIleVaIlePheThrV 15 alLeuLeuGInSerLeuCys 31 ValAlaValThrTyrValTyrPhe [hrAsnGluLeuLysGlnMetGlnAspLysTyrSerLysSerGlyI leAlaCysPheLeuLysGlu 20 61 AspAspSerTyrTrpAspProAsnAspGluGluSerMetAsnSerProCysTrpGlnValLysTrpGln LeuArgGlnLeuValArgLys 25 91 MetlleLeuArgThrSerGluGluL'hrlleSerThrValGnGluLysGnGnAsnIleSerProLeuVa ArgGluArgGlyProGin 121 30 ArgValAlaAlaHisIleThrGlyl'hrArgGlyArgSerAsnThrLeuSerSerProAsnSerLysAsnGl uLysAlaLeuGlyArgLys 151 IleAsnSerTrpGluSerSerArgSerGlyHisSerPheLeuSerAsnLeuHisLeuArgAsnGlyGluLe 35 uValIleHisGIuLysGly 181 PheTyrTyrIleTyrSerGlnThrl'yrPheArgPheGInGluGlulleLysGluAsnThrLysAsnAspL ysGlnMetValGlnTyrIle 40 211 TyrLysTyrThrSerTyrProAspProlleLeuLeuMetLysSerAlaArgAsnSerCysTrpSerLysAs pAlaGluTyrGlyLeuTyr 45 241 SerlleTyrGlnGlyGlyIlePheGluLeuLysGluAsnAspArgllePheValSerValThrAsnGluHis LeulleAspMetAspHis 271 GluAlaSerPhePheGlyAlaPheLeuValGly (SEQ ID NO:606) 178 3500906_1 (GHMatters) P77907.AU.I Apo-2L Sequence of Amino Acids 114-281 5 VRERGPQRVA AHITGTRGRS NTLSSPNSKN EKALGRKINS WESSRSGHSF LSNLHLRNGE LVIHEKGFYY IYSQTYFRFQ EEIKENTKND KQMVQYIYKY TSYPDPILLM KSARNSCWSK DAEYGLYSIY QGGIFELKEN DRIFVSVTNE HLIDMDHEAS FFGAFLVG (SEQ ID NO: 607) 10 179 3500906_1 (GHMtter) P77907.AU.1

Claims

2. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDR-H l comprises an amino acid sequence G-F-XI-1-X2-X3-X4-X5-1-H (SEQ ID NO:22), wherein G is position 26 and XI is position 28 according to the Kabat 30 numbering system; wherein Xl is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; 180 35698501 (GHMatters) P77907.AU.1 3-Aug-12 (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G (SEQ ID NO:23), wherein Xl is position 50 according to the Kabat numbering system; wherein X l is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; .wherein X5 is 5 selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 X9-XIO-X1I1-X12-X13-X14-X15-X16-X17-X18-X19-D-Y (SEQ ID NO:24), wherein XI is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI-X6 are selected from a pool of amino acids in a molar ratio of 50% Y, 25% S, D and 25% G; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a molar ratio of 50% Y, 25% S, and 25% G, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from I, M, L, and F, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOs:558-574,578,581 and 401
506. 5 3. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence G-F-X I -I-X2-X3-X4-X5-1-H (SEQ ID NO:22), wherein G is position 26 and Xl is position 28 according to the Kabat 0 numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G (SEQ ID NO:23),wherein XI is position 50 according to the Kabat 25 numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X I -X2-X3-X4-X5-X6-X7-X8 X9-XI0-X 11-X1 2-X1 3-X14-XI 5-XI6-X1 7-X1 8-XI9-D-Y (SEQ ID NO:26), wherein XI is 30 position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI-X6 are selected from a pool of amino acids in a molar ratio of 25% Y, 50% S, and 25% R; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a molar ratio of 25% Y, 50% S, and 25% R, or are not present; wherein X18 is 181 35696501 (GHMatters) P77907AU.1 3-Aug-12 selected from G and A; and wherein X 19 is selected from 1, M, L, and F, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOs:558-574, 578, 581 and 40 1-506. 5 4. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH I1 comprises an amino acid sequence G-F-X1-I-X2-X3-X4-X5-I-H (SEQ ID NO:22), wherein G is position 26 and XI is position 28 according to the Kabat numbering system; wherein Xl is selected from Y and S; wherein X2 is selected from Y and 0 S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G (SEQ ID NO:23),wherein Xl is position 50 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and 5 S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: XI-X2-X3-X4-X5-X6-X7-X8 X9-X1O-XI 1-X12-X13-X14-X15-X16-X17-X18-X19-D-Y (SEQ ID NO:27), wherein XI is position 95 according to the Kabat numbering system, and wherein the amino acids at each of 0 positions X1-X6 are selected from a pool of amino acids in a molar ratio of 38% Y, 25% S, 25% G, and 12% R; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a molar ratio of 38% Y, 25% S, 25% G, and 12% R, or are not present; wherein X18 is selected from G and A; and wherein X 19 is selected from I, M, L, and F, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOs:558 25 574, 578, 581 and 401-506. 5. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRHI comprises an amino acid sequence G-F-XI-I-X2-X3-X4-X5-I-H 30 (SEQ ID NO:22), wherein G is position 26 and X l is position 28 according to the Kabat numbering system; wherein Xl is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; 182 3569650_1 (GHMatters) P77907.AU.I 3-Aug-12 (ii) CDRI-12 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G (SEQ ID NO:23),wherein X1 is position 50 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is 5 selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 X9-X10-XI I-XI2-X13-X14-XI5-X16-X17-X18-XI9-D-Y (SEQ ID NO:28), wherein X1 is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI-X6 are selected from a pool of amino acids in a molar ratio of 20% Y, 26% S, 0 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W, or are not present; wherein X18 is selected from G and A; and wherein X19 is 5 selected from I, M, L, and F, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOs:558-574, 578, 581 and 401-506. 6. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: !0 (i) CDRH1 comprises amino acid sequence G-F-XI-I-X2-X3-X4-X5-I-H (SEQ ID NO:22); wherein XI is at position 28 according to Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; and X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence of XI-I-X2-P-X3-X4-G-X5-T 25 X6-Y-A-D-S-V-K-G (SEQ ID NO:23); wherein XI is at amino acid position 50 according to Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; X5 is selected from S and Y; and X6 is selected from S and Y; and (iii) CDRH3 comprises an amino acid sequence XI-X2-X3-X4-X5-X6-X7-D-Y 30 (SEQ ID NO:582), wherein X1 is at amino acid position 95 according to Kabat numbering and is selected from Y and R; X2 is selected from Y, S and R; X3 is selected from S, G, Y and H; X4 is selected from S, G, Y and R; X5 is selected from G and A; X6 is selected from F, M, L, and A; and X7 is selected from F, M, and L or is missing. 183 35698501 (GHMatters) P77907.AU.1 3-Aug-12 7. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH I comprises amino acid sequence G-F-XI-I-X2-X3-X4-X5-1-H (SEQ 5 ID NO:22); wherein XI is at position 28 according to Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; and X5 is selected from S and Y; (ii) CDRH2 comprises an amino acid sequence of X l-I-X2-P-X3-X4-G-X5-T X6-Y-A-D-S-V-K-G (SEQ ID NO:23); wherein XI is at amino acid position 50 according to 0 Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; and X5 is selected from S and Y; and (iii) CDRH3 comprises an amino acid sequence XI-X2-X3-X4-X5-X6-X7-X8-D Y (SEQ ID NO:583), wherein XI is at amino acid position 95 according to Kabat numbering and is selected from Y, S and G; X2 is selected from Y, S, G, R, A, and M; X3 is selected 5 from G, Y, S and R; X4 is selected from G, Y and F; X5 is selected from Y, S, N, and G; X6 is selected from Y, R, H and W; X7 is selected from G and A; and X8 is selected from F, M, L and I. 8. A polypeptide comprising an immunoglobulin heavy chain variable domain, !0 wherein: (i) CDRH1 comprises amino acid sequence G-F-XI-I-X2-X3-X4-X5-1-H (SEQ ID NO:22); wherein XI is at position 28 according to Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; and X5 is selected from S and Y; 25 (ii) CDRH2 comprises an amino acid sequence of XI -I-X2-P-X3-X4-G-X5-T X6-Y-A-D-S-V-K-G (SEQ ID NO:23); wherein XI is at amino acid position 50 according to Kabat numbering and is selected from S and Y; X2 is selected from S and Y; X3 is selected from S and Y; X4 is selected from S and Y; and X5 is selected from S and Y; and (iii) CDRH3 comprises an amino acid sequence of XI-X2-X3-X4-X5-X6-X7-X8 30 X9-XlO-XI l-X12-XI 3-X1 4-Xl 5-X16-XI 7-X1 8-X19-D-Y (SEQ ID NO:584), wherein XI is at amino acid position 95 and is selected from Y, S, R, G and E; X2 is selected from Y, S, R and G; X3 is selected from S, Y, G and W; X4 is selected from S, Y, G and Q; X5 is selected from G, Y and S; X6 is selected from G, Y, S, R and V; X7 is selected from S, Y, G 184 35696501 (GHMalters) P77907.AU I 3-Aug-12 and R; X8 is selected from Y, S, G, R, P and V; X9 is selected from G, A,Y, S and R; X10 is selected from M, F, G, Y, S and R; XII is selected from A,Y, S, G and R or is not present; X12 is selected from I, M, F, L, A, G, S, Y, R, and T or is not present; X13 is selected from F, M, L, G, A, Y, T, and S or is not present; X14 is selected from L, M, F, I, G, Y, A, and T 5 or is not present; X15 is selected from M, L, Y, G and R or is not present; X16 is selected from Y and G or is not present; X17 is selected from R, M, and G or is not present; X18 is selected from P and A or is not present; and Xl 9 is L or is not present, wherein CDR-H3 comprises an amino acid sequence selected from SEQ ID NOS: 429-506. 0 9. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH1 comprises an amino acid sequence G-F-XI-I-X2-X3-X4-X5-1-H, wherein G is position 26 and Xl is position 28 according to the Kabat numbering system; and wherein X l -X5 are naturally occurring amino acids other than cysteine; 5 (ii) CDRH2 comprises an amino acid sequence: X6-i-X7-P-X8-X9-G-X10-T X l -Y-A-D-S-V-K-G, wherein X6 is position 50 according to the Kabat numbering system, and wherein X6-X I I are naturally occurring amino acids other than cysteine; and (iii) CDRI-13 comprises an amino acid sequence: X12-X I 3-XI4-XI5-X16-(X17)" Xl 8-X I 9-D-Y, wherein X12 is position 95 according to the Kabat numbering system, and 0 wherein n is a suitable number that would retain the functional activity of the heavy chain variable domain, and wherein X12-X19 are naturally occurring amino acids other than cysteine, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOS: 429-506. 25 10. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH I comprises an amino acid sequence G-F-X I -I-X2-X3-X4-X5-1-H, wherein G is position 26 and XI is position 28 according to the Kabat numbering system; wherein X1 is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is 30 selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G, wherein XI is position 50 according to the Kabat numbering system; 185 356850_1 (GHMatters) P77907 AUI 3-Aug-12 wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: XI-X2-X3-X4-X5-X6-X7-X8 5 X9-XIO-XI 1-X12-X13-X14-X15-X16-X17-X18-X19, wherein Xl is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI-X17 are selected from S and one of A, C, F, G, I, L, N, P, R, T, W, or Y, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from F, L, I, and M, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOS:870-896. 0 11. A polypeptide comprising an immunoglobulin heavy chain variable domain, wherein: (i) CDRH I comprises an amino acid sequence G-F-X1-I-X2-X3-X4-X5-I-H, wherein G is position 26 and X l is position 28 according to the Kabat numbering system; 5 wherein the amino acid at each of positions XI-X5 is selected from S and one of Y, W, R, or F; (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5-T-X6 Y-A-D-S-V-K-G, wherein XI is position 50 according to the Kabat numbering system; wherein the amino acid at each of positions XI-X6 is selected from S and one of Y, W, R, 0 or F; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 X9-X I 0-XI 1 -X12-X 1 3-XI4-X I 5-X I 6-X 1 7-X 1 8-X19, wherein XI is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions X1 -X 17 are selected from S and one of Y, W, R, or F, or are not present; wherein X 18 is selected from G 25 and A; and wherein X19 is selected from F, L, I, and M, wherein CDRH3 comprises an amino acid sequence selected from SEQ ID NOS: 978-1004. 12. A library comprising a plurality of the polypeptide of any one of claims I to 11, and wherein the library has at least I x 104 distinct antibody variable domain sequences. 30 13. A method of generating a composition comprising a plurality of polypeptides comprising: (a) generating a plurality of polypeptides comprising: 186 3589850_1 (GHMatters) P77907.AU I 3-Aug-12 (i) CDRH I comprising an amino acid sequence G-F-Xl-I-X2-X3-X4-X5 I-H, wherein G is position 26 and X l is position 28 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is 5 selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprising an amino acid sequence: XI-I-X2-P-X3-X4-G X5-T-X6-Y-A-D-S-V-K-G, wherein X1 is position 50 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is 0 selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprising an amino acid sequence: XI-X2-X3-X4-X5-X6 X7-X8-X9-XIO-XI I -X12-X13-X14-X15-X16-X17-XI 8-X19, wherein XI is position 95 according to the Kabat numbering system, and wherein the amino 5 acids at each of positions X I-X17 are selected from S and one of A, C, F, G, I, L, N, P, R, T, W, or Y, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from F, L, I, and M. 14. A method of generating a composition comprising a plurality of polypeptides ,0 comprising: (a) generating a plurality of polypeptides comprising: (i) CDRH I comprises an amino acid sequence G-F-XI-I-X2-X3-X4-X5 I-H, wherein G is position 26 and XI is position 28 according to the Kabat numbering system; wherein the amino acid at each of positions XI-X5 is 25 selected from S and one of Y, W, R, or F; (ii) CDRH2 comprises an amino acid sequence: X1-I-X2-P-X3-X4-G-X5 T-X6-Y-A-D-S-V-K-G, wherein X l is position 50 according to the Kabat numbering system; wherein the amino acid at each of positions X1-X6 is selected from S and one of Y, W, R, or F; and 30 (iii) CDRH3 comprises an amino acid sequence: XI-X2-X3-X4-X5-X6 X7-X8-X9-X 10-X 11 -X1 2-X 1 3-X 14-XI 5-XI 6-X I 7-X1 8-X19, wherein XI is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI-X17 are selected from S and one of Y, W, R, or 187 3569650_1 (GHMatters) P77907.AU-1 3-Aug-12 F, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from F, L, I, and M. 15. A method of generating a composition comprising a plurality of polypeptides 5 comprising: (a) generating a plurality of polypeptides comprising: (i) CDRHI comprises an amino acid sequence G-F-XI-1-X2-X3-X4-X5 I-H, wherein G is position 26 and Xl is position 28 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is 0 selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprises an amino acid sequence: XI-I-X2-P-X3-X4-G-X5 T-X6-Y-A-D-S-V-K-G, wherein XI is position 50 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is 5 selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprises an amino acid sequence: X1-X2-X3-X4-X5-X6 X7-X8-X9-XIO-X 11-X12-XI3-X14-XI5-XI6-X17-XI8-XI9-D-Y, wherein .0 XI is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions X1-X6 are selected from a pool of amino acids in a molar ratio of 50% Y, 25% S, and 25% G; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a molar ratio of 50% Y, 25% S, and 25% G, or are not present; wherein X18 is 25 selected from G and A; and wherein X19 is selected from I, M, L, and F. 16. A method of generating a composition comprising a plurality of polypeptides comprising: (a) generating a plurality of polypeptides comprising: 30 (i) CDRHI comprising an amino acid sequence G-F-XI-I-X2-X3-X4-X5 I-H, wherein G is position 26 and XI is position 28 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is 188 3589650_1 (GHMalter) P77907 AU.1 3-Aug-12 selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprising an amino acid sequence: XI-I-X2-P-X3-X4-G X5-T-X6-Y-A-D-S-V-K-G, wherein Xl is position 50 according to the Kabat 5 numbering system; wherein X l is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprising an amino acid sequence: XI-X2-X3-X4-X5-X6 0 X7-X8-X9-X 10-X 1 I 2-X 13-X I 4-XI 5-X 1 6-X 1 7-XI 8-XI 9-D-Y, wherein X I is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions XI -X6 are selected from a pool of amino acids in a molar ratio of 25% Y, 50% S, and 25% R; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino acids in a 5 molar ratio of 25% Y, 50% S, and 25% R, or are not present; wherein Xl18 is selected from G and A; and wherein X19 is selected from I, M, L, and F. 17. A method of generating a composition comprising a plurality of polypeptides comprising: !0 (a) generating a plurality of polypeptides comprising: (i) CDRH1 comprising an amino acid sequence G-F-XI-1-X2-X3-X4-X5 I-H, wherein G is position 26 and Xl is position 28 according to the Kabat numbering system; wherein X l is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is 25 selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprising an amino acid sequence: XI-I-X2-P-X3-X4-G X5-T-X6-Y-A-D-S-V-K-G, wherein XI is position 50 according to the Kabat numbering system; wherein XI is selected from Y and S; wherein X2 is selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is 30 selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprising an amino acid sequence: XI-X2-X3-X4-X5-X6 X7-X8-X9-X1 0-X 11 -X 2-XI 3-X14-X I 5-XI 6-X 1 7-X1 8-X I 9-D-Y, wherein 189 35e98501 (GHMatters) P77907AU.1 3-Aug-12 Xl is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions X1 -X6 are selected from a pool of amino acids in a molar ratio of 38% Y, 25% S, 25% G, and 12% R; wherein the amino acids at each of positions X7-X17 are selected from a pool of amino 5 acids in a molar ratio of 38% Y, 25% S, 25% G, and 12% R, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from I, M, L, and F. 18. A method of generating a composition comprising a plurality of polypeptides 0 comprising: (a) generating a plurality of polypeptides comprising: (i) CDRHI comprising an amino acid sequence G-F-XI-I-X2-X3-X4-X5 I-H, wherein G is position 26 and Xl is position 28 according to the Kabat numbering system; wherein X l is selected from Y and S; wherein X2 is 5 selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; and wherein X5 is selected from Y and S; (ii) CDRH2 comprising an amino acid sequence: XI-I-X2-P-X3-X4-G X5-T-X6-Y-A-D-S-V-K-G, wherein Xl is position 50 according to the Kabat numbering system; wherein Xl is selected from Y and S; wherein X2 is 0 selected from Y and S; wherein X3 is selected from Y and S; wherein X4 is selected from Y and S; wherein X5 is selected from Y and S; and wherein X6 is selected from Y and S; and (iii) CDRH3 comprising an amino acid sequence: XI-X2-X3-X4-X5-X6 X7-X8-X9-X1O-X 11-Xl 2-XI 3-X14-X15-X16-X I 7-XI 8-XI9-D-Y, wherein 25 X I is position 95 according to the Kabat numbering system, and wherein the amino acids at each of positions X1-X6 are selected from a pool of amino acids in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1% I, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1% W; wherein the amino acids at each of positions X7-X17 are selected from 30 a pool of amino acids in a molar ratio of 20% Y, 26% S, 26% G, 13% R, 1% A, 1% D, 1% E, 1% F, 1% H, 1%1, 1% K, 1% L, 1% M, 1% N, 1% P, 1% Q, 1% T, 1% V, and 1 % W, or are not present; wherein X18 is selected from G and A; and wherein X19 is selected from I, M, L, and F. 190 3559650 1 (GHMetters) P77907.AUI 3-Aug. 12 19. A method of selecting for an antigen binding variable domain that binds to a target antigen from a library of antibody variable domains comprising: (a) contacting the library of claim 12 with a target antigen; 5 (b) separating one or more polypeptides that specifically bind to the target antigen from polypeptides that do not specifically bind to the target antigen, recovering the one or more polypeptides that specifically bind to the target antigen, and incubating the one or more polypeptides that specifically bind to the target antigen in a series of solutions comprising decreasing amounts of the target antigen in a concentration from 0 about 0.1 nM to about 1000 nM; and (c) selecting the one or more polypeptides that specifically bind to the target antigen and that can bind to the lowest concentration of the target antigen or that have an affinity of about 0.1 nM to about 200 nM. 5 20. A method of selecting for a polypeptide that binds to a target antigen from a library of polypeptides, comprising: (a) isolating one or more polypeptides that specifically bind to the target antigen by contacting a library comprising a plurality of polypeptides of any one of claims 1 to 1I with an immobilized target antigen under conditions suitable for binding; 0 (b) separating the one or more polypeptides that specifically bind to the target antigen from polypeptides that do not specifically bind to the target antigen, and recovering the one or more polypeptides that specifically bind to the target antigen to obtain a subpopulation enriched for the one or more polypeptides that specifically bind to the target antigen; and 25 (c) optionally, repeating steps (a)-(b) at least twice, each repetition using the subpopulation enriched for the one or more polypeptides that specifically bind to the target antigen obtained from the previous round of selection. 21. A method of isolating one or more polypeptides that specifically bind to a 30 target antigen with high affinity, comprising: (a) contacting a library comprising a plurality of polypeptides of any one of claims 1 to 11 with a target antigen at a concentration of at least about 0.1 nM to 191 3589850_1 (GHMalters) P77907 AU.1 3-Aug-12 about 1000 nM to isolate one or more polypeptides that specifically bind to the target antigen; (b) recovering the one or more polypeptides that specifically bind to the target antigen from the target antigen to obtain a subpopulation enriched for the one or more 5 polypeptides that specifically bind to the target antigen; and (c) optionally repeating steps (a) and (b) at least twice, each repetition using the subpopulation obtained from the previous round of selection and using a decreased concentration of target antigen from that used in the previous round to isolate one or more polypeptides that bind specifically to the target antigen at the lowest 0 concentration of target antigen. 22. The polypeptide of any one of claims I to I I that binds human HER2 or that binds human and murine DR5. 5 23. The polypeptide of any one of claims I to I1 or 22, wherein the polypeptide is an antibody. 24. The polypeptide of any one of claims I to 11, the library of claim 12, or the method of any one of claims 13 to 21, substantially as hereinbefore described with reference 0 to the examples and figures. 192 355 O501 (GHMatters) P77907.AU I 3-Aug-12