FIELD OF INVENTION

[0001] This invention is directed to a process of CDR-grafting and chain repairing for generating a library of humanized antibodies and isolating the antibodies from said library.

BACKGROUND OF THE INVENTION

[0002] One of the most promising therapeutics against human cancer and other diseases is that of antibody therapy. Typically, antibodies are derived from non-human sources, whose application in humans necessitates reduction of their immunogenicity when administered to human subjects. Various methodologies have been developed to address this issue, including the generation of chimeric antibodies, so-called "humanization" of antibodies and generation of antibodies in transgenic mice expressing human immunoglobulin genes.

[0003] Some methods for humanizing antibodies rely on CDR-grafting of the donor from a non-human source onto the most similar human acceptor antibody framework, however, the results in general show a decrease and even complete loss of binding activity in comparison to the parent antibody. Other methods include specific mutations in the antibody molecule, in an attempt to balance diminished immunogenicity with preservation of structural integrity and ultimately antibody affinity. While in some cases, the methods have produced "humanized" antibodies, which exhibit reasonable binding affinity and minimal immunogenicity, arrival at such molecules is via time- and labor-intensive procedures, and is not systematically applicable.

SUMMARY OF THE INVENTION

[0004] In one embodiment, the invention provides a process of CDR grafting and chain repairing for preparing a humanized antibody library, said process comprising the steps of grafting the three CDRs from the heavy chain (VH) of a murine antibody to a similar human heavy chain framework. In another embodiment, the process of CDR grafting and chain repairing for preparing a humanized antibody library comprises combining the newly grafted human heavy chain sequence with a human light chain (VL) library to prepare a library of humanized antibodies, wherein, In another embodiment, the humanized antibody is selected from said library for its original antigen binding. [0005] In one embodiment, the invention provides a process of CDR grafting and chain repairing for preparing a humanized antibody library, said process comprising the steps of: compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a similar human heavy chain sequence to that of a murine antibody, wherein said similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region (FR) residues from both human and said murine antibody; grafting said murine antibody's three CDRs onto said similar human heavy chain framework region; and, combining said CDR-grafted human heavy chain sequence with a human light chain (VL) chain library to generate a library of CDR-grafted humanized antibodies.

[0006] In one embodiment, the invention provides a library of CDR-grafted humanized antibodies of known specificity prepared by a process of CDR grafting and chain repairing comprising the steps of: compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a similar human heavy chain (VH) sequence to that of a murine antibody, wherein said similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region (FR) residues from both human and said murine antibody; grafting said murine antibody's CDRs onto said similar human heavy chain framework region; and, combining said CDR-grafted human heavy chain sequence with a human light chain (VL) chain library to generate a library of CDR-grafted humanized antibodies.

[0007] In one embodiment, the invention provides a method of identifying a humanized antibody having wild-type or optimized affinity to a known target, said method comprising the steps of: compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a similar human heavy chain (VH) sequence to that of a murine antibody; wherein said similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region residues from both human and a said parental non-human antibody. In another embodiment, the method of identifying a humanized antibody having wild-type or optimized affinity to a known target comprises grafting said murine antibody's CDRs onto said similar human heavy chain framework region. In yet another embodiment, the method of identifying a humanized antibody having wild-type or optimized affinity to a known target further comprises combining said CDR-grafted human heavy chain sequence with a human light chain (VL) library to generate a library of CDR-grafted humanized antibodies. In another embodiment, the invention provides a method of identifying a humanized antibody having wild-type or optimized affinity to a known target further comprises determining a respective binding affinity for a target for each of the humanized antibodies in the library of CDR-grafted antibodies and, identifying an antibody in the library of CDR-grafted antibodies having wild-type or optimized binding affinity for the target.

[0008] In one embodiment, the invention provides a kit for preparing a library of humanized antibodies. In another embodiment, the kit comprises a library of polynucleotides encoding a human light chain sequence. In another embodiment, the kit comprises a polynucleotide encoding a human heavy chain sequence that is similar to the sequence of a murine antibody whose grafting of its CDR is desired and, a polynucleotide encoding a murine antibody fragment comprising a complementarity determining region (CDR) region, whose grafting onto said human heavy chain similar sequence is desired and reagents for grafting said nucleic acid encoding a murine antibody fragment comprising complementarity determining region (CDR) regions onto said human heavy chain similar sequence.

[0009] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[00010] The invention will be better understood from a reading of the following detailed description taken in conjunction with the drawings in which like reference designators are used to designate like elements, and in which:

[00011] Figure 1. Design of the grafted VH chains. Two libraries for monoclonal antibody C225 (225Vc and 225Vd) were constructed. Those libraries contained large human VL chain library paired with the single VH chain in which two different set of CDRs (Kabat CDRs in Vc and Chothia CDRs in Vd) were grafted. Black letters: Common in mouse and human; Bold letters: mouse type residue; Bold underlined letters: human type residue; Shaded letters: grafted CDR. [00012] Figure 2. EGFR binding of the humanized antibodies isolated from 225Vc library and their VL chain sequence similarities, (a) Binding to EGFR by various phage clones recovered after two round of selection. Phage supernatants obtained from overnight culture (without dilution: gray bars; with 1/64 dilution by PBS: black bars) were added to 96-well plates coated with EGFR. Nine most active clones were highlighted by shaded letters, (b) Protein sequence diversity of the selected VL chains. Closest germlines were shown by; Bold letters: kV3-15; Italic: kV3-l l; Bold underlined: kV3D-15; Bold italic: kV3-20, Underlined: kVl-5. Nine most active clones were highlighted by shaded letters. The clone marked by # (2F2) was shown that Fab expression level on the phage was low. The clone marked by $ (2E6) had an unexpected mutation, G44S, in the VH chain.

[00013] Figure 3. VL chain sequence of the nine best humanized antibodies isolated from 225Vc library. Bold letters: Identical; Bold Italic letters: Conservative; Italic letters: Similar; Regular letters: Non-similar; Shaded letters: CDR.

[00014] Figure 4. The activity of the humanized Fab fragments obtained from 225Vc library, (a) EGFR binding, (b) Inhibition of EGF-induced EGFR phosphorylation, (c) Inhibition of cell proliferation.

[00015] Figure 5. EGFR binding by the VL chain selected from 225Vc library (clone Vc-2A6) paired with VH chain of Vd library or various Vc/Vd chimeric VH chains. Phage ELISA result showed that Vd-2A6 did not show any binding, but all chimeric VH chains were active. Compared to Vd which has Chothia CDRl and CDR2, F-CD (Kabat CDRl and Chothia CDR2) and F-DC (Chothia CDRl and Kabat CDR2) binds to EGFR, indicating grafting of one of Kabat defined CDRl and CDR2 retains antigen binding activity. The activity of F-DC was ~30-fold higher than that of F-CD, indicating CDR2 is more important for binding. The results also showed that the part of Kabat CDR2 recovers the activity. For example, gain of mouse type residues DTPFT at the positions H58 and 61-64 (C-DC) recovers the binding to some extend, and further mouse type mutation VaI at H50 (F-DC) improves the activity about 4-fold. Similarly, compared to Vc in which CDRs were both Kabat defined, the loss of mouse residues DTPFT at 58 and 61-64 (C-CD) decrease binding slightly (4-fold), and the further loss of VaI at H50 (F-CD) affect more strikingly (17-fold). DETAILED DESCRIPTION OF THE INVENTION

[00016] In one embodiment, the invention relates to a "grafting and shuffling" humanization strategy that has allowed the generation of multiple diverse humanized anti-EGFR Fabs of a mouse antibody, C225. Compared to the standard CDR grafting methods, this approach is simple, rapid and efficient. The new humanization library contains single acceptor human heavy chain grafted with donor CDRs of the parental mouse antibody combined with human light chain repertoires. In this method, the three CDRs from the VH chain of a non- human antibody are grafted to a single and similar human VH chain framework, combined with a human VL chain library, and selected for original antigen binding. This method overcomes the major problems often associated with the conventional rational humanization strategy, in that the method does not need structural information of the antibody to be humanized, nor the repeated design-and-test cycles for making back mutations in order to restore the original affinity. Since the library contains a single VH sequence which contains the original mouse CDRs, the library size is largely reduced compared to the other empirical strategies. Two libraries were constructed and the humanized antibodies were successfully isolated from one of them. The best clone, 2A6, retained similar antigen binding affinity and the biological activity of the original mouse C225. The best humanized antibody exhibited only a 2-fold affinity loss when compared with the parental Fab, without further refinement by back mutation, however, the selection of the residues to be grafted is critical. Although CDR3 of the VH chain are often most important for the antigen binding, CDRl and CDR2 also have a contributory effect. Further, and in one embodiment, Kabat defined CDRs works completely in the methods provided herein but not Chothia defined CDRs.

[00017] In one embodiment, provided herein is a process of CDR grafting and chain repairing for preparing a humanized antibody library, said process comprises the steps of grafting the three CDRs from the heavy chain (VH) of a murine antibody to a similar human heavy chain framework. In another embodiment of the present invention, the human antibody framework is derived from one of or a combination of the heavy chain variable region subgroup III (VH III), VH I, VH II or from any other heavy chain variable region as will be understood by a skilled artisan. In another embodiment, the murine antibody is M225 or any other murine antibody available in the arts for use in the methods and compositions provided herein. In another embodiment, the similar human heavy chain framework is derived from a human antibody. In another embodiment, the similar human heavy chain framework sequence is selected from any one of SEQ ID NOs: 2-3. In another embodiment, the human antibody is AAB67785 (SEQ ID NO: 4) or any other human antibody available in the art for use in the methods and compositions provided herein. In another embodiment, the CDR residues are defined according to the Kabat definition. In yet another embodiment, the CDR residues are defined according to the Chothia or Enhanced Chothia definition as described below. It will be readily apparent to a skilled artisan that the methods described herein can be used to humanize any other antibodies.

[00018] In one embodiment, provided herein is a process of CDR grafting and chain repairing for preparing a humanized antibody library, said process comprises the steps of compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a single and similar human heavy chain sequence to that of a murine antibody, wherein, in another embodiment said similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region (FR) residues from both human and said murine antibody. In another embodiment, the process of CDR grafting and chain repairing for preparing a humanized antibody library further comprises grafting the murine antibody's three CDRs onto the similar human heavy chain framework region. In yet other embodiments, the process of CDR grafting and chain repairing for preparing a humanized antibody library further comprises combining the CDR-grafted human heavy chain sequence with a human light chain (VL) chain library to generate a library of CDR-grafted humanized antibodies. In another embodiment, provided herein is a human heavy chain framework region sequence similar to a parental murine antibody's framework region sequence, identified and prepared according to the process described herein.

[00019] In one embodiment, the most homologous human VH sequence to that of the original murine antibody are identified by searching against all non-redundant GenBank CDS databases by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). During the search, CDR residues in the KABAT definition are masked and only mouse FR residues are aligned with amino acid sequences of functional human antibodies. In another embodiment, AAB67785 was selected as human VH FRs for M225, because it has high sequence homology (amino acid identity = 67.8%; similarity = 89.7%), and belong to the same subgroup of the original VH (human heavy chain subgroup II).

[00020] In another embodiment, using the AAB67785 sequence, two humanized VH chains are designed, in which two different set of CDRs, as defined Kabat and Chothia definitions (Kabat et al., 1977; Chothia et al., 1989), respectively, are grafted to the human framework region (FR) (Fig. 1). The CDR-grafted VH chains are then used to create 225Vc and 225Vd libraries, respectively.

[00021] In another embodiment, percentage sequence "identity" refers to a number of identical residues in a pairwise alignment divided by the total number of aligned residues, including the gaps. In another embodiment, the term "similar" refers to a number of similar residues in a pairwise alignment divided by the total number of aligned residues, including the gaps. In another embodiment, these residues are ones that have side-chains that share similar biochemical properties, for example hydrophobicity, hydrophilicity, and the like. In another embodiment, identical residues are similar but the inverse is not true, therefore identity percentage is smaller than the similarity percentage for sequence pairwise alignments.

[00022] In another embodiment, the invention provides for humanized antibody library generation, and antibodies and functional fragments thereof, comprising a single heavy chain framework region (FR) templates onto which an appropriate complementarity determining region (CDR) is grafted followed by combining the grafted heavy chain with light chain repertoires to generate a humanized antibody library. In another embodiment, each antibody within the library comprises of the same human heavy chain framework gene sequence. In another embodiment, each antibody within the library consists of the same heavy chain gene framework sequence. In another embodiment, each antibody within the library comprises a different human light chain sequence. In another embodiment, the light chain (VL) variable region for the VL library is derived from the Kappa III (VK III) or from any other light chain variable region as will be appreciated by a skilled artisan. In another embodiment, each antibody within the library consists of a different human light chain sequence. In another embodiment each isolated antibody has a preserved desired affinity for a particular target, in a context, which in another embodiment, allows for minimal to no immune-mediated rejection of the molecule.

[00023] In another embodiment, the process of CDR grafting and chain repairing further comprises combining the newly grafted human heavy chain sequence with a human light chain (VL) library to prepare a library of humanized antibodies, wherein and in another embodiment, a humanized antibody is selected from the library for its original antigen binding. The invention further provides, in one embodiment, a human heavy chain framework for murine complementarity determining region (CDR) grafting. In another embodiment, each human heavy chain framework shares an overall sequence of more than 61 % with that of a murine antibody. In another embodiment, each human heavy chain framework shares an overall sequence of more than 71% with that of a murine antibody. In another embodiment, each human heavy chain framework shares an overall sequence of more than 61 % with that of a murine antibody. In another embodiment, each human heavy chain framework shares an overall sequence of more than 81% with that of a murine antibody. In another embodiment, each human heavy chain framework shares an overall sequence of more than 91% with that of a murine antibody. In another embodiment, each human heavy chain framework shares an overall sequence of more than 95% with that of a murine antibody. In another embodiment, the murine antibody is M225. This murine antibody is an EGFR blocking antibody. In another embodiment, the process further comprises selecting a murine monoclonal antibody of known specificity for optimization and grafting a complementarity determining region (CDR) of the murine monoclonal antibody onto a heavy chain framework that comprises a similar heavy chain framework sequence to that of the murine antibody's framework.

[00024] In one embodiment, the process of CDR grafting and chain repairing further provides an isolated humanized antibody or functional fragment thereof. In another embodiment, the fragment is an Fab fragment, an scFv fragment or any functional antibody fragment as will be understood by a skilled artisan. In another embodiment, the humanized antibody is derived from any one of clones 2A6, 2A4, 2El, 2C8, 2Cl 1, 2A10, 2C5, 2Fl 1, or 2G12, respectively corresponding to SEQ ID NOs: 6-14. In another embodiment, provided herein is a human light chain (VL) sequence identified from a clone selected from the sequence of any one of SEQ ID NOs: 6-14.

[00025] In another embodiment, a parental antibody is the antibody from which the CDR is derived, and can be viewed as a donor, while the various antibodies which will be included in the libraries of the invention, comprise human framework that receive the CDR-grafts described herein, and therefore may be viewed as acceptors. Grafting is accomplished, in another embodiment, by splicing a population of encoding parental nucleic acids for the donor CDR into a population of nucleic acids encoding for a human antibody acceptor variable region framework which contains species representing all desired amino acid residue changes within the CDR positions.

[00026] In one embodiment, the term "framework region" or "FR" are those variable domain residues other than the hypervariable region residues. The framework regions have been precisely defined. See, e.g., Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, National Institutes of Health, USA (5th ed. 1991). Each variable domain typically has four FRs identified as FRl, FR2, FR3 and FR4. In another embodiment, "FR" also refers to an antibody variable region comprising amino acid residues abutting or proximal to, but outside of the CDR regions i.e., regions which directly interact with the antigen, acting as the recognition element of the antibody molecule within the variable region of an antibody. In one embodiment, the term "framework region" is intended to mean each domain of the framework that is separated by the CDRs. In another embodiment, the sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The combined heavy and light chain framework regions of an antibody serve to position and align the CDRs for proper binding to the antigen.

[00027] In one embodiment, the term "CDR" or "complementarity determining region" refers to amino acid residues comprising non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. In another embodiment, the "CDR" is further defined using the Enhanced Chothia numbering scheme and the "Contact CDR" definition described herein. In another embodiment, the term "CDR" will comprise regions as described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987) and MacCallum et al., J. MoI. Biol. 262:732-745 (1996). The amino acids of the CDRs of the variable domains were initially defined by Kabat, based on sequence variability, to consist of amino acid residues 31-35B (Hl), 50-65 (H2), and 95-102 (H3) in the human heavy chain variable domain (VH) and amino acid residues 24-34 (Ll), 50-56 (L2), and 89-97 (L3) in the human light chain variable domain (VL), using Rabat's numbering system for amino acid residues of an antibody. See Kabat et al., sequences of proteins of immunological interest, US Dept. Health and Human Services, NIH, USA (5th ed. 1991). Chothia and Lesk, J. MoI. Biol. 196:901-917 (1987) presented another definition of the CDRs based on residues that included in the three-dimensional structural loops of the variable domain regions, which were found to be important in antigen binding activity. Chothia et al. defined the CDRs as consisting of amino acid residues 26- 32 (Hl), 52-56 (H2), and 95-102 (H3) in the human heavy chain variable domain (VH), and amino acid residues 24-34 (Ll), 50-56 (L2), and 89-97 (L3) in the human light chain variable domain (VL). Combining the CDR definitions of Kabat and Chothia, the CDRs consist of amino acid residues 26-35B (Hl), 50-65 (H2), and 95-102 (H3) in human VH and amino acid residues 24-34 (Ll), 50-56 (L2), and 89-97 (L3) in human VL, based on Kabat's numbering system.

[00028] Alteration of donor CDR amino acid positions is also considered an embodiment of the present invention, which can be necessary to produce humanized antibodies optimized for affinity to the original antigen as will be appreciated by a skilled artisan. Amino acid residues selected for alteration during binding affinity optimization are typically amino positions predicted to be relatively important for function. Criteria that can be used for identifying amino positions to be altered include, for example, conservation of amino acids among polypeptide subfamily members, conservation of binding affinity and/or avidity, the desirability of low to no immunogenicity to the host and knowledge that particular amino acids are predicted to be important in polypeptide conformation or structure, as described herein. [00029] In one embodiment, the term "donor" is intended to mean a parent antibody molecule or functional fragment thereof from which a portion is derived from, given or contributes to another antibody molecule or fragment thereof so as to confer either a functional characteristic of the parent molecule onto the receiving molecule. For the specific example of CDR grafting, the parent molecule from which the grafted CDRs are derived is a donor molecule. The donors CDRs confer binding affinity of the parent molecule onto the receiving molecule. Instead, it is sufficient that the donor is a separate and distinct molecule.

[00030] In another embodiment, the known target is a tumor antigen, whereby, in another embodiment, the tumor antigen is a cell surface antigen such as EGFR, IGFR, gp75, Flt-3, CD20, CD19, CD33, CD59, CD46, Fas, AFP, PDGFR, FGFR, gp-75, c-Met, CXCR4, CSFR, TGFR, c-Kit and VE-cadherin, RON, CA 125, CEA, CD19, CD20, CD44, T cell receptor alpha/beta, CD45, VEGFR, GD ₂ , GD ₃ , GMl, GM2, Her-2/Neu, Ep-CAM (KSA), endothelin receptor, IL-2 receptor, Lewis-Y, Lewis-X (CD 15), melanoma-associated proteoglycan MCSP, PSA, Transferrin Receptor, NKG2D and its ligands, MICA and MICB, the extracellular domain of the receptors listed herein or any other cell surface tumor antigen receptor or any other cell surface tumor antigen or fragment thereof.

[00031] In another embodiment, the term "antibody" refers to intact molecules as well as functional fragments thereof, such as Fab, F(ab') ₂ , and Fv that are capable of specifically interacting with a desired target. In another embodiment, the antibody fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) F(ab') ₂ , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') ₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

[00032] In another embodiment, the term "antibody" or "fragments thereof comprise "humanized forms" of antibodies. In another embodiment, the term "humanized forms of antibodies" refers to non-human (e.g. murine) antibodies, which are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[00033] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[00034] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-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., Nature, 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoL Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].

[00035] In one embodiment, provided herein is a method of identifying a humanized antibody having wild- type or optimized affinity to a known target, said method comprising the steps of: compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a similar human heavy chain (VH) sequence to that of a murine antibody, wherein, in another embodiment, the similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region residues from both human and a said parental non-human antibody. In another embodiment, the method of identifying a humanized antibody having wild-type or optimized affinity to a known target, further comprises the steps of grafting the murine antibody's CDRs onto said similar human heavy chain framework region. In another embodiment, the method of identifying a humanized antibody having wild-type or optimized affinity to a known target further comprises the step of combining said CDR- grafted human heavy chain sequence with a human light chain (VL) chain library to generate a library of CDR- grafted humanized antibodies. In yet another embodiment, the method of identifying a humanized antibody having wild-type or optimized affinity to a known target, further comprises the steps of determining a respective binding affinity for a target for each of said antibodies in said library of CDR-grafted antibodies and identifying an antibody in said library of CDR-grafted antibodies having wild-type or optimized binding affinity for said target. In another embodiment, provided herein is a humanized antibody or fragment thereof optimized for affinity to a known target.

[00036] Antibodies with known specificity whose affinity is desired to be optimized by the methods of this invention may be constructed by any means known in the art. For example, monoclonal antibodies may be produced in a number of ways, including using the hybridoma method (e.g. as described by Kohler et al., Nature, 256: 495, 1975, herein incorporated by reference), or by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567, herein incorporated by reference). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. In one embodiment, suitable myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody- producing cells, and are sensitive to a medium such as HAT medium. For example, murine myeloma lines, such as those derived from MOPC-21 and MPC-I l mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (e.g., Kozbor, J. Immunol., 133: 3001 (1984), herein incorporated by reference).

[00037] In one embodiment, to test efficiency of humanization, 225Vc (SEQ ID NO: 2) and 225Vd (SEQ ID NO: 3) libraries are used for selection against EGFR by phage display. In another embodiment, after each round of selection, recovered phage clones are picked randomly and isolated phages are subjected to phage ELISA for EGFR binding activity. In another embodiment, humanized antibody clones are successfully isolated from 225Vc library as demonstrated herein below in Example 2.

[00038] In one embodiment, the term "binds" or "binding" or grammatical equivalents, refer to the compositions having affinity for each other. "Specific binding" is where the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding can be distinguished from non-specific when the dissociation constant (K _D ) is less than about IxIO ^"5 M or less than about IxIO ^"6 M or IxIO ^"7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like. Appropriate controls can be used to distinguish between "specific" and "non-specific" binding.

[00039] Additional selection systems may be used, including, but not limited to the hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al., Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and the adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes. Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047 (1988)); and ODC (ornithine decarboxylase), which confers resistance to the ornithine decarboxylase inhibitor, 2- (difluoromethyl)-DL-omi thine, DFMO (McConlogue (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed.).

[00040] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[00041] DNA or nucleic acid encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[00042] In another embodiment, the term "nucleic acid" refers to a polynucleotide or to oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA) or mimetic thereof. The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double- stranded polynucleotides. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non- naturally- occurring portions, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[00043] As will be appreciated by one skilled in the art, a fragment or derivative of a nucleic acid sequence or gene that encodes for a protein or peptide can still function in the same manner as the entire, wild type gene or sequence. Likewise, forms of nucleic acid sequences can have variations as compared to wild type sequences, nevertheless encoding a protein or peptide, or fragments thereof, retaining wild type function exhibiting the same biological effect, despite these variations.

[00044] The nucleic acids of the present invention can be produced by any synthetic or recombinant process such as is well known in the art. Nucleic acids according to the invention can further be modified to alter biophysical or biological properties by means of techniques known in the art. For example, the nucleic acid can be modified to increase its stability against nucleases (e.g., "end-capping"), or to modify its lipophilicity, solubility, or binding affinity to complementary sequences.

[00045] Methods for modifying nucleic acids to achieve specific purposes are disclosed in the art, for example, in Sambrook et al. (1989). Moreover, the nucleic acid sequences of the invention can include one or more portions of nucleotide sequence that are non-coding for the protein of interest. The invention further provides DNA sequences which encode proteins similar to those encoded by sequences as described herein, but which differ in terms of their codon sequence due to the degeneracy of the genetic code or allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change), which may encode the proteins of the invention described herein, as well. Variations in the DNA sequences, which are caused by point mutations or by induced modifications (including insertion, deletion, and substitution) to enhance the activity, half-life or production of the polypeptides encoded thereby, are also encompassed in the invention.

[00046] In one embodiment, the isolated polypeptide of this invention includes modification to the original sequence of the native protein. "Modification" is to be understood as comprising non-native amino acid residues and sequences of such non-native residues, which have been introduced as a consequence or mutation of the native sequence (by either random or site-directed processes). [00047] In one embodiment, a humanized antibody is generated by the process described herein and is then purified or isolated after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is well known in the art, a variety of natural proteins bind antibodies, for example bacterial proteins A, G, and L, and these proteins may find use in the present invention for purification. Purification can often be enabled by a particular fusion partner. For example, proteins may be purified using glutathione resin if a GST fusion is employed, Ni ⁺² affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag- tag is used. For general guidance in suitable purification techniques, see Protein Purification: Principles and Practice, 3 ^rd Ed., Scopes, Springer- Verlag, N.Y., 1994.

[00048] In one embodiment the isolated polypeptide of this invention is a fragment of the native protein. In one embodiment, the term "fragment" refers to a protein or polypeptide that is shorter or comprises fewer amino acids than the full length protein or polypeptide. In another embodiment, a fragment refers to a nucleic acid that is shorter or comprises fewer nucleotides than the full length nucleic acid. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, the fragment of this invention is an intrasequential section of the protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional intrasequential section of the protein, peptide or nucleic acid. In another embodiment, the fragment is a functional intrasequential section within the protein, peptide or nucleic acid. In another embodiment, the fragment is an N-terminal functional fragment. In one embodiment, the fragment is a C-terminal functional fragment.

[00049] In another embodiment, a "variable region" when used in reference to an antibody or a heavy or light chain thereof is intended to mean the amino terminal portion of an antibody which confers antigen binding onto the molecule and which is not the constant region. The term is intended to include functional fragments thereof which maintain some or all of the binding function of the whole variable region. Therefore, the term "heteromeric variable region binding fragments" is intended to mean at least one heavy chain variable region and at least one light chain variable regions or functional fragments thereof assembled into a heteromeric complex. Heteromeric variable region binding fragments include, for example, functional fragments such as Fab, F(ab) ₂ , Fv, single chain Fv (scfv) and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. [00050] In one embodiment, provided herein is a library of CDR-grafted humanized antibodies of known specificity prepared by a process of CDR grafting and chain repairing comprising the steps of: compiling sequence information from a large non-redundant variable region dataset of human antibodies and selecting a similar human heavy chain (VH) sequence to that of a murine antibody, wherein, in another embodiment, the similar VH sequence is arrived at by masking the complementarity determining region (CDR) residues and aligning framework region (FR) residues from both human and said murine antibody. In another embodiment a library of CDR-grafted humanized antibodies of known specificity, prepared by a process of CDR grafting and chain repairing further comprises the steps of grafting said murine antibody's CDRs onto said similar human heavy chain framework region; and, combining said CDR-grafted human heavy chain sequence with a human light chain (VL) chain library to generate a library of CDR-grafted humanized antibodies.

[00051] In one embodiment, in constructing new libraries for humanization, the single grafted VH gene segment is cloned into the large Fab phage display library replacing the original diverse VH repertoire.

[00052] In another embodiment, genes encoding the grafted VH chain are synthesized commercially and subcloned into pUCminusMCS vector (Blue Heron Biotech) or any other suitable vector know in the art. In another embodiment, to attach CHl gene, the synthesized VH gene was amplified by PCR, and digested with B stEII at the C-terminus. In another embodiment, the CHl gene is amplified by PCR from pCESl phagemid vector, and digested with BstEII at the N-terminus. Subsequently, the synthesized VH and CHl genes are ligated together, and amplified by PCR again. After digestion with Ncol and Notl, VH-CHl fragment was cloned back to pCESl vector as Exemplified herein.

[00053] In one embodiment, for the source of the VL repertories is gathered from a human Fab phage display library in a suitable vector known in the art. In another embodiment, a phagemid is first digested with suitable enzymes, such as Sfil and Notl, then treated with antarctic phosphatase (NEB), and purified by agarose gel electrophoresis to remove VH-CHl gene fragment from the VL chain-containing backbone vector. In one embodiment, the grafted VH-CHl fragment described above is amplified by PCR from pCESl vector, digested with Sfil and Notl, and replaced VH gene in a library vector. Thus, in new libraries and in another embodiment, a single grafted VH is paired with 10 ⁷ -10 ⁸ VL repertories. In another embodiment, E. coli TGl cells (Stratagene) are then transformed with the new library phagemid by electroporation, and grown on ten large 2YT-AG plates (2YT medium plate containing 2% glucose and 100 μg/ml ampicillin). In yet another embodiment, small amount of transformants are used for titration to confirm library size. In another embodiment, after incubation overnight at 30 ⁰ C, all colonies on the plates are scraped into 25 ml 2YT-AG medium, mixed with 8 ml 80% glycerol, and stored at -80 ⁰ C as the library stock.

[00054] In one embodiment, a chimeric Vc/Vd heavy (VH) chain is generated as follows: four oligonucleotide primers are commercially synthesized (Invitrogen), which encode both strands of the center region of FR2 and CDR2 where the sequences are common in the VH genes of Vc and Vd. The N-terminus half VH genes including FR1-(1/2)FR2 or FR1-(1/2)CDR2, and the C-terminus half VH genes including (1/2)FR2-FR4 or (1/2)CDR2-FR4 are amplified by PCR. Subsequently, the chimeric VH genes are amplified by assembly PCR of N-terminus Vc fragment and C-terminus Vd fragment, or vice versa, using overlapping regions, and cloned back into the phagemid vector paired with the VL chain of the clone Vc-2A6 which are selected from Vc library for EGFR binding described above. It is to be understood by a skilled artisan that other methods known in the art can be used to arrive at a chimeric Vc/Vd VH chain.

[00055] In one embodiment, the library provided herein is a nucleic acid library or a phage display library or an oligopeptide library. In another embodiment, the process yields a Fab fragment library, a FR library, a VH library, a VL library, a VH and VL library, a CDR library or an ScFv fragment library. In one embodiment no structural information is required to prepare the humanized antibody library. According to this aspect of the invention, and in another embodiment, the invention provides a library of humanized antibodies of known specificity prepared according to a process of the invention. In one embodiment, for proof-of-concept of new "grafting and shuffling" humanization strategy, two libraries for C225 (225Vc-SEQ ID NO: 2 and 225Vd- SEQ ID NO: 3) were constructed. In another embodiment, those libraries contain large human VL chain library paired with the single VH chain in which two different set of CDRs are grafted, that is, Kabat CDRs in Vc and Chothia CDRs in Vd ( See Fig. 1).

[00056] In one embodiment, the term "library" refers to a repertoire of human light chain sequences that have been combined with a grafted human heavy chain in any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions at variable positions, a physical library comprising nucleic acids that encode the library sequences, or a physical library comprising the repertoire of human light chain sequences in combination with a grafted human heavy chain sequence, either in purified or unpurified form. Accordingly, there are a variety of techniques that may be used to efficiently generate libraries of the present invention as will be understood by a skilled artisan. Such methods include but are not limited to gene assembly methods, PCR-based methods and methods which use variations of PCR, ligase chain reaction-based methods, pooled oligo methods such as those used in synthetic shuffling, error- prone amplification methods and methods which use oligos with random mutations, classical site-directed mutagenesis methods, cassette mutagenesis, and other amplification and gene synthesis methods. As is known in the art, there are a variety of commercially available kits and methods for gene assembly, mutagenesis, vector subcloning, and the like, and such commercial products find use in generating the libraries provided herein.

[00057] In one embodiment, a library of grafted human immunoglobulin heavy chain sequences is prepared by using phage display to combine with a repertoire of human light chain sequences as exemplified in the examples section herein.

[00058] In one embodiment, a "phage display library" refers to a collection of phage (e.g., filamentous phage) wherein the phage expresses an external (typically heterologous) protein. The external protein is free to interact with (bind to) other moieties with which the phage are contacted. Each phage displaying an external protein is a "member" of the phage display library.

[00059] The term "filamentous phage" or "filamentous bacteriophage" refers to a viral particle capable of displaying a heterogeneous polypeptide on its surface. Although one skilled in the art will appreciate that a variety of bacteriophage may be employed in the present invention, in preferred embodiments the vector is, or is derived from, a filamentous bacteriophage, such as, for example, fl, fd, PfI, M13, etc. The filamentous phage may contain a selectable marker such as tetracycline (e.g., "fd-tet"). Various filamentous phage display systems are well known to those of skill in the art (see, e.g., Zacher et al. (1980) Gene 9: 127-140, Smith et al.(1985) Science 228: 1315-1317 (1985); and Parmley and Smith (1988) Gene 73: 305-318).

[00060] An assembly cell is a cell in which a nucleic acid can be packaged into a viral coat protein (capsid). Assembly cells may be infected with one or more different virus particles (e.g. a normal or debilitated phage and a helper phage) that individually or in combination direct packaging of a nucleic acid into a viral capsid.

[0006I] In another embodiment, the method of preparing a phage display humanized antibody library comprises in another embodiment, the steps of modifying a phagemid vector for cloning, assembling VH and VL chains, followed by sequence analysis, sequential cloning of VL and VH into the phagemid vector, and building a large size library.

[00062] In one embodiment, a phage library is created by inserting a library of a random oligonucleotide or a cDNA library encoding antibody fragment such as VL and VH into gene III of M 13 or fd phage. Each inserted gene is expressed at the N-terminal of the gene III product, a minor coat protein of the phage. As a result, peptide libraries that contain diverse peptides can be constructed. The phage library is then affinity screened against immobilized target molecule of interest, such as an antigen, and specifically bound phages are recovered and amplified by infection into Escherichia coli host cells. Typically, the target molecule of interest such as a receptor (e. g., polypeptide, carbohydrate, glycoprotein, nucleic acid) is immobilized by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled for screen plaques or colony lifts. This procedure is called biopanning.

[00063] In one embodiment, the phages comprising the humanized antibody library are rescued and panned against EGFR or any antigen such as, but not limited to those described herein. In another embodiment, best binders are selected and their sequence is analyzed. In another embodiment, the best binders can be cloned into a phagemid vector to build a large library comprising the humanized antibody library. In another embodiment, the phagemid of individual selected clones are used to transform a nonsuppressor E. coli cells, and the soluble antibody can be expressed and purified from the periplasmic extract. Following expression and in another embodiment, the binding kinetics of the isolated humanized antibody can be measured using a BIAcore biosensor which can be based on surface plasmon resonance (SPR), an optical phenomenon that enables detection of unlabeled interactants in real time.

[00064] In one embodiment, the parental antibody is an anti-EGFR antibody. In another embodiment, the anti- EGFR antibody is selected from Cetuximab (Erbitux), Panitumomab (Vectibix), Zalutumomab (HuMax- EGFR), Matuzumab (EMD72000), or an anti-EGFRvIII antibody.

[00065] In one embodiment of the invention, the anti-EGFR antibody is Cetuximab. In another embodiment, the anti-EGFR antibody is any antibody selected from the following list of antibodies or an Fab, ScFv, or functional fragment thereof, that are represented by accession numbers ABG27073, ABX79402, ABX79401, ABX79400, ABX79399, ABX79398. ABX79397, ABX79396, ABX79395, ABX79394, ABX79393, ABX79392.

[00066] In one embodiment, methods of identifying antibodies through their binding affinities or specificities are very well known in the art and include methods such as immunoprecipitation or an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Other well-known methods can be used to determine antibody binding affinities and these methods can be readily used, as will be understood by a skilled artisan. In that regard, the method of the present invention further comprises determining a respective binding affinity for a target for each of the antibodies in the library formed. In another embodiment the method further comprises identifying an antibody having the highest binding affinity for a known target. According to this aspect, and in one embodiment, this invention provides a humanized antibody optimized for affinity to a known target identified by the methods of this invention. Antibodies with known specificity are prepared and their affinity assessed. In one embodiment, the binding kinetics of various humanized Fabs is determined by BIAcore analysis (See Table 1 and Example 3 herein below).

[00067] Once an antibody of interest has been identified from a library, DNAs encoding the light and heavy chains of the antibody can be isolated by standard molecular biology techniques, such as by polymerase chain reaction (PCR) amplification of DNA from the display package (e.g., phage) isolated during the library screening process. Nucleotide sequences of antibody light and heavy chain genes from which oligonucleotide primers can be determined using methods known in the art and as described herein.

[00068] In one embodiment, provided herein is a method of down-regulating expression of an oncogenic receptor or protein on the cell surface, comprising the step of contacting said receptor with the humanized antibody produced by the methods provided herein.

[00069] In one embodiment, provided herein is a method of inhibiting EGF- stimulated phosphorylation. In another embodiment, provided herein is a method of inhibiting cell proliferation. In another embodiment, provided herein is a method of inhibiting EGF-stimulated phosphorylation and cell proliferation. In another embodiment, the method comprises the step of administering an effective amount of the humanized antibody provided herein. In another embodiment, the humanized antibody of the present invention is able to able to inhibit EGFR phosphorylation on tyrosine residues in intact cells (see Figure 4 herein below).

[00070] In another embodiment, provided herein is a method of blocking an interaction between a receptor and a ligand comprising contacting said receptor with the humanized antibody produced by the methods provided herein. In another embodiment, the method of blocking an interaction between a receptor and a ligand further comprises the step of administering proteolytic inhibitors, pharmaceutical carriers, diluents, and adjuvants.

[00071] In another embodiment, provided herein is a method of preventing formation of a tumor in a subject comprising the step of administering an effective amount of the humanized antibody provided herein. [00072] In one embodiment, provided herein is a method of treating a tumor in a subject, comprising the step of administering an effective amount of the humanized antibody provided herein.

[00073] In one embodiment, provided herein is a method of inhibiting or suppressing a tumor in a subject, comprising the step of administering an effective amount of the humanized antibody provided herein.

[00074] In another embodiment, provided herein is a method of ameliorating symptoms associated with a tumor growth in a subject, comprising the step of administering an effective amount of the humanized antibody provided herein.

[00075] In another embodiment, the method of down-regulating expression of an oncogenic receptor or protein on the cell surface further, and the method of blocking an interaction between a receptor and a ligand further comprise the step of co-administering, in combination (concomitantly or individually) proteolytic inhibitors pharmaceutical carriers, diluents, and adjuvants. In another embodiment, the method of preventing formation of a tumor in a subject, the method of inhibiting or suppressing a tumor in a subject, and the method of ameliorating symptoms associated with a tumor growth in a subject further comprise the step of coadministering, in combination (concomitantly or individually) proteolytic inhibitors pharmaceutical carriers, diluents, and adjuvants. In yet another embodiment, the method of suppressing a tumor in a subject, and of treating a tumor in a subject further comprise the step of co-administering, in combination (concomitantly or individually) proteolytic inhibitors pharmaceutical carriers, diluents, and adjuvants.

[00076] The first step in the mitogenic stimulation of epithelial cells is the specific binding of epidermal growth factor (EGF) to a membrane glycoprotein known as the epidermal growth factor receptor (EGF receptor) Carpenter et al. (1979) Annual Review Biochem., Vol.: 48, pages 193-216. The EGF receptor is composed of 1186 amino acids which are divided into an extra-cellular portion of 621 residues and a cytoplasmic portion of 542 residues connected by a single hydrophobic trans-membrane segment of 23 residues described in Ulrich et al. (1986) Nature, Vol.: 309, pates 418-425. The external portion of the EGF receptor can be subdivided into four domains. The domain III, residues 333 to 460, which is connected by two cysteine domains, contains the EGF binding site of the receptor shown by Lax et al. (1988) MoI. and Cell Biol. Vol.: 8 pages 1831 to 1834. The binding of EGF to domain III leads to the initiation of pleiotropic responses leading to DNA synthesis and cell proliferation. [00077] Various types of human tumor cells show overexpression of the EGF receptor. For example, the cancerous cells of bladder tumors have been shown to have a relatively large population of EGF receptors described in Neal et al. (1985) Lancet, Vol.: 1 pages 366-367. The influence of EGF receptor density on the biological behavior of cancer cells may be mediated by the interaction of the receptor with its ligands- namely, EGF or transforming growth factor— α (TGF-α). In the majority of cells, when EGF binds to a specific region of the EGF receptor, the cell is mitogenically simulated. Other tumor cells, such as A431 cells, are not mitogenically stimulated by the binding of EGF to its receptor. Nevertheless, the tumor A413 is inhibited in nude mice by binding monoclonal antibodies to the epidermal growth factor receptor of the tumorous cells as shown by Masui et al. (1984) Cancer Res. Vol.: 44, pages 1002-1007. EP 0 359 282 describes an antibody specifically binding and inhibiting the growth of human tumors cells. In 1993 Naramura et al (1993) Cancer Immunol. Immunotherapy Vol.: 37, 343-349 describes the antibody Cetuximab or C225, which recognizes and binds to the EGF receptor. The problem with antibodies to coagulate and to aggregate is solved in WO 2003/007988.

[00078] There is a strong medical need for a medicament to effectively treat cancer, in particular head and neck cancer, non-small cell lung cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, and/or breast cancer. The present invention makes available novel and effective medicaments, which are suitable for the treatment of cancer, in particular head and neck cancer, non-small cell lung cancer, prostate cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric cancer, and/or breast cancer.

[00079] In one embodiment, the polypeptide or antibodies provided herein are "biologically active", meaning they are able to exert the biological action or an enhanced action of their corresponding parental antibodies even after modification, in particular in binding to the target antigen, inhibiting binding of ligands to receptors, further in terms of modulation, in particular inhibition of antigen-mediated signal transduction and prophylaxis or therapy of antigen-mediated diseases. The term "biologically active", when used in reference to any of the biologically active agents described herein also refers to the agent's ability to modulate the immune response in a manner that can lead to a preventive, diagnostic, or therapeutic effect as will be understood by a skilled artisan. In another embodiment, agents that can be used to achieve this biological activity include but are not limited to a cytokine, an enzyme, a chemokine, a radioisotope, an enzymatically active toxin, or a chemotherapeutic agent, as will be understood by a skilled artisan. [00080] In yet another embodiment, the polypeptides or antibodies provided herein are conjugated to a receptor for utilization in tumor pretargeting wherein the antibody-receptor or polypeptide-receptor conjugate is administered to the subject, 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 to a cytotoxic agent (e.g. a radionucleotide). In an alternate embodiment, the polypeptides of antibodies are conjugated or operably linked to an enzyme in order to employ Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used by conjugating or operably linking the antibody or Fc fusion to a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent) to an active anti-cancer drug. The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form. Enzymes that are useful in the method of this invention include but are not limited to alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D- alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate- cleaving enzymes such as β-galactosidase and neuramimidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with -α-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, for example, Massey, 1987, Nature 328: 457-458). Polypeptide/antibody-abzyme conjugates can be prepared for delivery of the abzyme to a tumor cell population. Other additional modifications of the modified molecules provided herein are contemplated herein. For example, the polypeptide/antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[00081] In another embodiment, the antibody/polypeptide provided herein is administered with one or more immunomodulatory agents or biologically active agents. In another embodiment, the biologically active compound or agent is an immunomodulatory agent. Such agents may increase or decrease production of one or more cytokines, up- or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells. Immunomodulatory agents include but are not limited to: non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g. glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidineicosanoids such as prostaglandins, thromboxanes, and leukotrienes; as well as topical steroids such as anthralin, calcipotriene, clobetasol, and tazarotene); cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-IO; cytokine, chemokine, or receptor antagonists including antibodies, soluble receptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CDI l, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFN-α, IFN-β, IFN-γ, IFNAR, IgE, IL-I, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA- 1, LFA-3, MHC, selectins, TGF-β, TNF-α, TNF-β, TNF-Rl, T-cell receptor, including Enbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab); heterologous anti-lymphocyte globulin; other immunomodulatory molecules such as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypic antibodies for MHC binding peptides and MHC fragments, azathioprine, brequinar, bromocryptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamides (e.g. leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulfasasazine.

[00082] In an alternate embodiment, antibodies of the present invention are administered with a cytokine. By "cytokine" as used herein is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet- growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, beta, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-I, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-I l, IL-12; IL-15, a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[00083] In one embodiment, the biologically active compound or agent provided in the methods and compositions herein is a chemotherapeutic or other cytotoxic agent. In another embodiment, the chemotherapeutic or cytotoxic agent is administered as a prodrug. The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, for example Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382; and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.): 247-267, Humana Press, 1985. The prodrugs that may find use with the compositions and methods as provided herein include but are not limited to phosphate-containing prodrugs, thiophosphate- containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide- containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use with the antibodies/polypeptides of the compositions and methods provided herein include but are not limited to any of the aforementioned chemotherapeutic agents.

[00084] In one embodiment, any combination of the antibody/polypeptide with the biological active agents specified above, i.e., a cytokine, an enzyme, a chemokine, a radioisotope, an enzymatically active toxin, or a chemotherapeutic agent can be applied. In another embodiment, the antibody/polypeptide can be operably- linked with the biologically active agent and used in the methods described herein or antibody/polypeptide provided herein can merely be used in combination with the biologically active agents, in a manner in which both are administered separately (i.e. - not conjugated) to achieve the desired preventive, diagnostic, or therapeutic effect. [00085] The pharmacokinetics (PK) of the antibodies of the invention can be studied in a variety of animal systems, with the most relevant being non-human primates such as the cynomolgus, rhesus monkeys. Single or repeated i.v./s.c. administrations over a dose range of 6000-fold (0.05-300 mg/kg) can be evaluated for the half-life (days to weeks) using plasma concentration and clearance as well as volume of distribution at a steady state and level of systemic absorbance can be measured. Examples of such parameters of measurement generally include maximum observed plasma concentration (Cmax), the time to reach Cmax (Tmax), the area under the plasma concentration-time curve from time 0 to infinity [AUC(0-inf] and apparent elimination half- life (Tm). Additional measured parameters could include compartmental analysis of concentration-time data obtained following i.v. administration and bioavailability. Examples of pharmacological/toxicological studies using cynomolgus have been established for Rituxan® and Zevalin® in which monoclonal antibodies to CD20 are cross-reactive. Biodistribution, dosimetry (for radiolabled antibodies), and PK studies can also be done in rodent models. Such studies would evaluate tolerance at all doses administered, toxicity to local tissues, localization to rodent xenograft animal models, depletion of target cells (e.g. CD20 positive cells).

[00086] In one embodiment, an antibody of the present invention is administered to a patient having a disease involving inappropriate expression of a target antigen, a protein or other molecule. Within the scope of the present invention this is meant to include diseases and disorders characterized by aberrant proteins, due for example to alterations in the amount of a protein present, protein localization, posttranslational modification, conformational state, the presence of a mutant or pathogen protein, etc. Similarly, the disease or disorder may be characterized by alterations molecules including but not limited to polysaccharides and gangliosides. An overabundance may be due to any cause, including but not limited to overexpression at the molecular level, prolonged or accumulated appearance at the site of action, or increased activity of a protein relative to normal. Included within this definition are diseases and disorders characterized by a reduction of a protein. This reduction may be due to any cause, including but not limited to reduced expression at the molecular level, shortened or reduced appearance at the site of action, mutant forms of a protein, or decreased activity of a protein relative to normal. Such an overabundance or reduction of a protein can be measured relative to normal expression, appearance, or activity of a protein, and said measurement may play an important role in the development and/or clinical testing of the antibodies of the present invention.

[00087] In one embodiment, the term "cancer" and "cancerous" refer to or describe, in one embodiment, the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

[00088] More particular examples of such cancers include hematologic malignancies, such as non-Hodgkin's lymphomas (NHL). NHL cancers include but are not limited to Burkitt's lymphoma (BL), small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL) and lymphoplasmacytic leukemia (LPL), extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B cell lymphoma, mediastinal large cell lymphoma, intravascular large cell lymphoma, primary effusion lymphoma, precursor B -lymphoblastic leukemia/lymphoma, precursor T- and NK-cells lymphoma (precursor T lymphoblastic lymphoma, blastic NK cell lymphoma), tumors of the mature T and NK cells, including peripheral T-cell lymphoma and leukemia (PTL), adult T-cell leukemia/T-cell lymphomas and large granular lymphocytic leukemia, T-cell chronic lymphocytic leukemia/prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK- cell leukemia, extranodal T-/NK cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, anaplastic large cell lymphoma (ALCL), angiocetric and angioimmunoblastic T-cell lymphoma, mycosis fungoides/Sezary syndrome, and cutaneous T-cell lymphoma (CTCL). Other cancers that may be treatable by the antibodies of the invention include but are not limited to Hodgkin's lymphoma, tumors of lymphocyte precursor cells, including B-cell acute lymphoblastic leukemia/lymphoma (B-ALL), and T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), thymoma, Langerhans cell histocytosis, multiple myeloma, myeloid neoplasias such as acute myelogenous leukemias (AML), including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes, and chronic myeloproliferative disorders (MDS), including chronic myelogenous leukemia (CML). Other cancers that may be treatable by the antibodies of the invention include but are not limited to tumors of the central nervous system such as glioma, glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma; solid tumors of the head and neck (e.g. nasopharyngeal cancer, salivary gland carcinoma, and esophageal cancer), lung (e.g. small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), digestive system (e.g. gastric or stomach cancer including gastrointestinal cancer, cancer of the bile duct or biliary tract, colon cancer, rectal cancer, colorectal cancer, and anal carcinoma), reproductive system (e.g. testicular, penile, or prostate cancer, uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer), skin (e.g. melanoma, basal cell carcinoma, squamous cell cancer, actinic keratosis), liver (e.g. liver cancer, hepatic carcinoma, hepatocellular cancer, and hepatoma), bone (e.g. osteoclastoma, and osteolytic bone cancers) additional tissues and organs (e.g. pancreatic cancer, bladder cancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer of the peritoneum, and Kaposi's sarcoma), and tumors of the vascular system (e.g. angiosarcoma and hemagiopericytoma).

[00089] In one embodiment, provided herein is a chimeric humanized heavy chain molecule comprising CDR-grafts from a murine antibody, wherein each heavy chain sequence from said chimeric humanized heavy chain is obtained from the two diverse libraries generated by the process of CDR-grafting and chain repairing provided herein. In another embodiment, the chimeric humanized heavy chain molecule CDR regions are defined using the Kabat and Chothia definitions. In another embodiment, the chimeric humanized heavy chain molecule of is F-CD, F-DC, C-DC, C-CD, or F-CD as each are defined herein below in example 5. In another embodiment, the chimeric humanized heavy chain molecule is further combined with a human light chain to generate a modified antibody molecule comprising said chimeric humanized heavy chain sequence. In another embodiment, the chimeric humanized heavy chain molecule comprises a unique CDR region sequence in at least a single CDR sequence that enhances binding to a known target, wherein the known target is a tumor antigen as described herein.

[00090] In one embodiment, the humanized antibodies or chimeric heavy chains provided herein can be variants of each other. A "variant" of a polypeptide or protein, in one embodiment, refers to an amino acid sequence that is altered with respect to the referenced polypeptide or protein by one or more amino acids. In the present invention, a variant of a polypeptide retains the antibody-binding property of the referenced protein. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). A variant may also have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological reactivity may be found using computer programs well known in the art, for example, DNASTAR software.

[00091] In one embodiment, provided herein is a method of determining the CDR residues critical for binding to a know target such as a tumor antigen as described herein, comprising the step of testing the chimeric humanized heavy chain molecule for binding to the tumor antigen. [00092] In another embodiment, the term "acceptor" is intended to mean an antibody heavy chain (VH) comprising a framework region (FR) which is to receive the donated portion from the parent or donor antibody molecule. An acceptor is therefore imparted, as with the functional characteristic of the donated portion of the parent molecule. For the specific example of CDR grafting, the receiving molecule for which the CDRs are grafted is an acceptor molecule. The acceptor antibody molecule or fragment is imparted with the binding affinity of the donor CDRs or parent molecule.

[00093] In one embodiment, an isolated polypeptide or antibody provided herein may comprise a derivate of a polypeptide of this invention, "derivative" is to be understood as referring, In another embodiment, to less than the full-length portion of the native sequence of the protein in question. In another embodiment, a "derivative" may further comprise (at its termini and/or within said sequence itself) non-native sequences, i.e. sequences which do not form part of the native protein in question. The term "derivative" also includes within its scope molecular species produced by conjugating chemical groups to the amino residue side chains of the native proteins or fragments thereof, wherein said chemical groups do not form part of the naturally- occurring amino acid residues present in said native proteins.

[00094] In one embodiment, the polypeptide antibody or variable region thereof of this invention comprises an amino acid substitution. In one embodiment, the amino acid substitution is conservative. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In another embodiment, the amino acid substitution may not be conservative which may result in enhanced activity of the mutated polypeptide compared to the native polypeptide.

[00095] In one embodiment, the term "polypeptide" refers a humanized antibody prepared by the process of CDR grafting and chain repairing provided herein. In another embodiment the term refers to a chimeric heavy chain molecule as provided herein. In another embodiment, the term refers to a chimeric heavy chain molecule combined with a human light chain as provided herein. The polypeptides or humanized antibodies of this invention can be produced by any synthetic or recombinant process as will be understood by a skilled artisan. Polypeptides can further be modified to alter biophysical or biological properties by means of techniques known in the art. For example, the polypeptide can be modified to increase its stability against proteases, or to modify its lipophilicity, solubility, or binding affinity to its native receptor.

[00096] In one embodiment, compiling sequence information involves assembling such information regarding multiple human antibodies of known specificity, and availing such information in a central location.

[00097] In one embodiment, the term "compiling" refers to a systematic storage of sequence information regarding antibody regions such as variable heavy and light chains that comprise complementarity determining regions and framework regions as provided herein. The systematic storage of sequence information of the invention provided herein makes use of any appropriate storage media, for example, a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as magnetic/optical storage media. The systematic storage of sequence information further makes use of any appropriate accessible database, software or any electronic/computerized/internet-based means of storing, analyzing, and/or retrieving, in a usable manner, sequence information as described herein or as it is available in the existing art. It is to be understood that any means of storage of such sequence information known in the art, cataloguing of such information, and arrangement of such information known in the art, in a manner facilitating analysis of such information by any means, for example by ranking such information based on statistical evaluation thereof, by grouping such information based on similarity of certain standards, or other annotations which facilitate the construction of libraries as herein described and as understood to the skilled artisan based thereupon, is to be considered as part of this invention.

[00098] In another embodiment, the term "compiling" refers to assembling sequence information derived from other sources and imported as is, i.e. by straight catalogue of results from probes of known databases. In another embodiment, the term refers to the preservation of a context and content that is readily accessible, in a format that can be easily probed, and assessed and subjected to mathematical and statistical calculations. In another embodiment, such context and content may comprise the ability to conduct index searches, keyword searches, or compile information from known sequence software programs or databases, for example DNASTAR software, IgBLAST, GenBank, BLAST2SEQ, PSI-BLAST, NCBI, or any additional database. The additional database can specifically contain arranged antibody information relating to sequence information of CDRs and FRs relating to the process of CDR grafting and chain repairing as described herein, and is to be considered as contemplated for use. Further, by "compiling" it is meant that the sequence information is obtained thereby ranking, indexing or sorting other information input by an end-user, including sequence information input by hand or by other means known in the art, and may include information prior to statistical or other analysis or following such analysis or both. The term "compiling" also will distinguish between the statistical or other analysis known in the art, such that the user can constantly update the databases and libraries thereby with new information as it becomes available, or refine such information based on new selection criteria in order to yield the libraries/polypeptides/antibodies/antibody fragments provided herein.

[00099] In one embodiment, the term "sequence information" refers to compiling nucleotide sequences (reflecting natural or synthetic DNA or RNA) encoding the FRs or amino acid sequences comprising the FRs. The sequence information can be complete, such as nucleic acid or protein sequences, or partial, such as positions of given nucleotides or amino acids within the DNA/RNA or protein, or fragments, respectively. In one embodiment, such sequence information is compiled from any available source, including available gene and/or amino acid sequence databases, for example Genbank, Genecards, Swiss-prot, Geneatlas or any existing database, or any database which may be assembled over time, which comprises such information, as will be appreciated by the skilled artisan. In another embodiment, compiling sequence information may also include ranking or sorting such information, and reflect scoring of such information, including inputting such information in a format, where such scoring reflects compiling "sorted" information, or In another embodiment, compiling reflects preparation of pre-sorted and post sorted information, such that compiling may occur over time, with the ability to continuously update such information and expand the humanized antibody libraries of this invention. One skilled in the art will appreciate that the use of sequence information may curb the introduction of substitutions that are potentially deleterious to antibody structure.

[000100] In another embodiment, the term "sequence alignment" refers arrangement of a primary sequence (DNA, RNA, or amino acid) to identify regions of similarity or identity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. In another embodiment, aligned sequences of nucleotide or amino acid residues are represented as rows within a matrix, and compiling includes storage of such matrices. In another embodiment, gaps are inserted between residues for purposes of arriving at greater sequence similarity over a longer stretch of residues in a particular sequence, as will be appreciated by the skilled artisan. [000101] Sequence alignment methods that can be used to achieve the desired sequence alignment include in another embodiment, but are not solely restricted to pair- wise alignment methods or multiple- sequence alignment methods, as will be understood by a skilled artisan. Sequence alignments can be stored in a wide variety of text-based file formats. In one embodiment, this is achieved by converting in certain embodiments, any format, for example a FASTA or GenBank, SwissProt, Entrez and EMBL format, using conversion programs and programming packages such as, READSEQ, EMBOSS and BioPerl, BioRuby. It is to be understood that a skilled artisan can convert, modify, score, update and/or store the sequences as necessary using any program or storage media, as will be appreciated by the skilled artisan. In another embodiment, the sequence alignment is scored using a method described herein or any method available in the art, for example BLOSUM (for BLOcks Substitution Matrix). In another embodiment, BLOSUM gives a score for each pair of amino acids based on how likely we will observe such a pair in alignments of truly conserved blocks of amino acids. A higher score indicates that such a pair of amino acids are often seen to be aligned to each other when we align functionally similar proteins with each other.

[000102] In another embodiment, the term "sequence alignment" includes use of any program or method, as understood by a skilled artisan, that is used to perform nucleic acid or amino acid sequence alignments to yield results that can be readily probed, assessed and subjected to mathematical and statistical calculations. In one embodiment, methods for sequence or alignment are well known in the art, and include alignments based on sequence homology, as described herein.

[000103] In one embodiment, the term "homology," "homolog" or "homologous" may refer to sequence identity, or sequence similarity, or to functional identity. By using the term "homology" and the other like forms, it is to be understood that any molecule, whether nucleic acid or peptide, that functions similarly, and/or contains sequence identity, and/or is conserved structurally so that it approximates the reference sequence, is to be considered as part of this invention. In another embodiment, the terms "homology", "homologue" or "homologous", in any instance, indicate that the sequence referred to, whether an amino acid sequence, or a nucleic acid sequence, exhibits at least 60% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 70% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 80% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 97% or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 97% - 100% correspondence to the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 100% correspondence to the indicated sequence. Similarly, in one embodiment, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined. Accordingly and in one embodiment, the term "non-homologous" refers to an amino acid sequence or nucleic acid sequence that exhibits no more than 60% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 55% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 54-44% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 43-34% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 33-24% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 24-14% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 13-044% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 3-0.1% correspondence with the indicated sequence.

[000104] In one embodiment, pair- wise alignment methods are used to find the best- matching piecewise (local) or global alignments of two query sequences. In another embodiment, multiple pairwise alignments are used.

[000105] In one embodiment, the heavy chain sequences of a human antibody of the invention are aligned separately with the sequences of a parental murine antibody to identify the sequences with the highest homology. Nucleic acid or Polypeptide homology for any nucleic acid or polypeptide sequence may be determined by algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via methods well known to one skilled in the art. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis. [000106] In another embodiment, "dot-matrix" methods refer to an alignment approach which implicitly produces a family of alignments for individual sequence regions and these methods can are also available for use in accordance with the invention. In another embodiment, a dot-matrix plot is constructed, comprising construction of a two-dimensional matrix having "dots" placed at any point of character match (see "Alignment of Pairs of Sequences," Chapter 3, in Bioinformatics: Sequence and Genome Analysis, 2nd edition, by David W. Mount. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.) In another embodiment, the size or intensity of the dot varies as a function of the degree of similarity of the two characters, to accommodate conservative substitutions. The dot plots of very closely related sequences appear as a single line along the matrix's main diagonal. In one embodiment, dot plots can also be used to assess repetitiveness in a single sequence. This sequence can be plotted against itself and regions that share significant similarities will appear as lines off the main diagonal. In another embodiment, this effect occurs when a protein consists of Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group multiple similar structural domains.

[000107] For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids may be grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non- conservative substitutions constitute exchanging a member of one of these classes for a member of another.

[000108] In another embodiment, "dynamic programming" methods can be applied to produce global alignments via the Needleman-Wunsch algorithm (Needleman SB, Wunsch CD. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". J MoI Biol 48 (3): 443- 53. PMID 5420325), and local alignments via the Smith-Waterman algorithm. Dynamic programming can be useful in aligning nucleotide to protein sequences. The framesearch method produces a series of global or local pairwise alignments between a query nucleotide sequence and a search set of protein sequences, or vice versa. In another embodiment, frameshift offset by an arbitrary number of nucleotides makes the method useful for sequences containing large numbers of indels, which can be very difficult to align with other methods. In another embodiment, the method requires large amounts of computing power or a system whose architecture is specialized for dynamic programming. The BLAST and EMBOSS suites provide basic tools for creating translated alignments (though some of these approaches take advantage of side-effects of sequence searching capabilities of the tools). In another embodiment, commercial sources of such programs are available for use in accordance with the invention, such as FrameSearch, distributed as part of the Accelrys GCG package, and Open Source software such as Genewise.

[000109] In another embodiment, the dynamic programming method provides an optimal alignment given a particular scoring function, which in another embodiment, is best applied in alignments of pairwise comparisons.

[000110] In another embodiment, "word methods" may be used, which comprise their implementation in database search tools such as the FASTA and the BLAST family Mount DM (2004. Bioinformatics: Sequence and Genome Analysis 2nd ed.. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY. ISBN 0- 87969-608-7). In one embodiment, word methods identify a series of short, nonoverlapping subsequences ("words") in the query sequence that are then matched to candidate database sequences. The relative positions of the word in the two sequences being compared are subtracted to obtain an offset; this will indicate a region of alignment if multiple distinct words produce the same offset. Only if this region is detected do these methods apply more sensitive alignment criteria; thus, many unnecessary comparisons with sequences of no appreciable similarity are eliminated. In the FASTA method, the user defines a value k to use as the word length with which to search the database.

[000111] In selecting a heavy chain comprising a framework region (e.g. human germline), in another embodiment, a number of criteria may be employed (although employing selection criteria is not necessary to understand or practice the present invention). For example, one may select a heavy chain that is known to be less immunogenic for a particular host (e.g. human). An additional criteria that may be used (e.g., for humans) in order to minimize the risk of immunogenicity, is to eliminate human genes that are not-functional or that are infrequently used in the human population (i.e. select a heavy chain that is frequently used in the human population). One could also select a heavy chain derived from population analysis from a variety of ethnic populations, e.g. arrive at a heavy chain commonly found in European Caucasian, different FR backbones in Africans, provided that in all cases the heavy chain sequence has a high similarity with the framework of the parental antibody sequence (e.g. murine antibody). By selecting these frameworks, when armed with the invention provided herein, a skilled artisan can graft the CDRs from the parental antibodies and proceed to combine the grafted human heavy chain sequence with a human light chain library in order to generate humanized antibody libraries to then isolate individual antibodies with the desired characteristics, affinity, and specificity.

[000112] A variety of selection methods are known in the art that may find use in the present invention for screening protein libraries. These include but are not limited to phage display (Phage display of peptides and proteins: a laboratory manual, Kay et al., 1996, Academic Press, San Diego, Calif., 1996; Lowman et al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science 228: 1315-1317) and its derivatives such as selective phage infection (Malmborg et al., 1997, J MoI Biol 273:544-551), selectively infective phage (Krebber et al., 1997, J MoI Biol 268:619-630), and delayed infectivity panning (Benhar et al., 2000, J MoI Biol 301:893-904), cell surface display (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399) such as display on bacteria (Georgiou et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993, Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; June et al., 1998, Nat Biotechnol 16:576-80), yeast (Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder & Wittrup, 1997, Nat Biotechnol 15:553-557), and mammalian cells (Whitehorn et al., 1995, Bio/technology 13:1215-1219), as well as in vitro display technologies (Amstutz et al., 2001, Curr Opin Biotechnol 12:400-405) such as polysome display (Mattheakis et al., 1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes et al., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts & Szostak, 1997, Proc Natl Acad Sci USA 94: 12297- 12302; Nemoto et al., 1997, FEBS Lett 414:405-408), and ribosome-inactivation display system (Zhou et al., 2002, J Am Chem Soc 124, 538-543).

[000113] Other selection methods that may find use in the present invention include methods that do not rely on display, such as in vivo methods including but not limited to periplasmic expression and cytometric screening (Chen et al., 2001, Nat Biotechnol 19:537-542), the protein fragment complementation assay (Johnsson & Varshavsky, 1994, Proc Natl Acad Sci USA 91: 10340-10344; Pelletier et al., 1998, Proc Natl Acad Sci USA 95: 12141-12146), and the yeast two hybrid screen (Fields & Song, 1989, Nature 340:245-246) used in selection mode (Visintin et al, 1999, Proc Natl Acad Sci USA 96: 11723-11728). In an alternate embodiment, selection is enabled by a fusion partner that binds to a specific sequence on the expression vector, thus linking covalently or noncovalently the fusion partner and associated variant library member with the nucleic acid that encodes them. For example, U.S. Ser. No. 09/642,574; U.S. Ser. No. 10/080,376; U.S. Ser. No. 09/792,630; U.S. Ser. No. 10/023,208; U.S. Ser. No. 09/792,626; U.S. Ser. No. 10/082,671; U.S. Ser. No. 09/953,351; U.S. Ser. No. 10/097,100; U.S. Ser. No. 60/366,658; PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a fusion partner and technique that may find use in the present invention. In an alternative embodiment, in vivo selection can occur if expression of the protein imparts some growth, reproduction, or survival advantage to the cell.

[000114] In another embodiment, recombinant antibody libraries can be expressed on the surface of yeast cells or bacterial cells. Methods for preparing and screening libraries expressed on the surface of yeast cells are described further in International Application Publication No. WO 99/36569. Methods for preparing and screening libraries expressed on the surface of bacterial cells are described further in U.S. Pat. No. 6,699,658.

[000115] In one embodiment, a library is screened using one or more cell-based or in vitro assays. For such assays, antibodies, purified or unpurified, are typically added exogenously such that cells are exposed to individual variants or groups of variants belonging to a library. These assays are typically, but not always, based on the biology of the ability of the antibody to bind to antigen and mediate some biochemical event, for example effector functions like cellular lysis, phagocytosis, ligand/receptor binding inhibition, inhibition of growth and/or proliferation, apoptosis, etc. Such assays often involve monitoring the response of cells to antibody, for example cell survival, cell death, cellular phagocytosis, cell lysis, change in cellular morphology, or transcriptional activation such as cellular expression of a natural gene or reporter gene. For example, such assays may measure the ability of antibodies to elicit ADCC, ADCP, or CDC. For some assays additional cells or components, that is in addition to the target cells, may need to be added, for example serum complement, or effector cells such as peripheral blood monocytes (PBMCs), NK cells, macrophages, and the like. Such additional cells may be from any organism, e.g., humans, mice, rats, rabbits, monkeys, etc. Crosslinked or monomelic antibodies may cause apoptosis of certain cell lines expressing the antibody's target antigen, or they may mediate attack on target cells by immune cells which have been added to the assay. Methods for monitoring cell death or viability are known in the art, and include the use of dyes, fluorophores, immunochemical, cytochemical, and radioactive reagents. For example, caspase assays or annexin-flourconjugates may enable apoptosis to be measured, and uptake or release of radioactive substrates (e.g. Chromium-51 release assays) or the metabolic reduction of fluorescent dyes such as alamar blue may enable cell growth, proliferation, or activation to be monitored. In one embodiment, the DELFIA EuTDA- based cytotoxicity assay (Perkin Elmer, MA) is used. Alternatively, dead or damaged target cells may be monitored by measuring the release of one or more natural intracellular proteins, for example lactate dehydrogenase. Transcriptional activation may also serve as a method for assaying function in cell-based assays. In this case, response may be monitored by assaying for natural genes or proteins which may be upregulated or down-regulated, for example the release of certain interleukins may be measured, or alternatively readout may be via a luciferase or GFP-reporter construct. Cell-based assays may also involve the measure of morphological changes of cells as a response to the presence of an antibody. Cell types for such assays may be prokaryotic or eukaryotic, and a variety of cell lines that are known in the art may be employed. Alternatively, cell-based screens are performed using cells that have been transformed or transfected with nucleic acids encoding the antibodies.

[000116] Introduction of nucleic acid encoding the heavy or light chains or parental CDRs provided herein into target cells can be carried out by conventional methods known in the art such as osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques. For example, a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene- vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. A nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[000117] The use of liposomes for introducing various compositions into cells, including nucleic acids, is known to those skilled in the art (see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282). A carrier comprising a natural polymer, or a derivative or a hydrolysate of a natural polymer, described in WO 94/20078 and U.S. Pat. No. 6,096,291, is suitable for mucosal delivery of molecules, such as polypeptides and polynucleotides. Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Pat. No. 5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat. No. 5,459,127). Accordingly, viral and non-viral vector means of delivery into cells or tissue, in vitro, in vivo and ex vivo are included.

[000118] In one embodiment, nucleotide sequences can be operably linked, i.e., positioned, to ensure the functioning of an expression control sequence. These expression constructs are typically replicable in the cells either as episomes or as integral parts of the cell's chromosomal DNA, and may contain appropriate origins of replication for the respective prokaryotic strain employed for expression. Commonly, expression constructs contain selection markers, such as for example, tetracycline resistance, ampicillin resistance, kanamycin resistance or chloramphenicol resistance, facilitating detection and/or selection of those bacterial cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Pat. No. 4,704,362). These markers, however, are not exclusionary, and numerous others may be employed, as known to those skilled in the art. Indeed, in a preferred embodiment of the present invention expression constructs contain both positive and ne "g&a _" tive selection markers.

[000119] Similarly reporter genes may be incorporated within expression constructs to facilitate identification of transcribed products. Accordingly, in one embodiment of the present invention, reporter genes utilized are selected from the group consisting of β-galactosidase, chloramphenicol acetyl transferase, luciferase and a fluorescent protein.

[000120] Prokaryotic promoter sequences regulate expression of the encoded polynucleotide sequences, and in another embodiment of the present invention, are operably linked to polynucleotides encoding the polypeptides of this invention. In additional embodiments of the present invention, these promoters are either constitutive or inducible, and provide a means of high and low levels of expression of the polypeptides of this invention, and in another embodiment, for regulated expression of multiple polypeptides of the invention, which in another embodiment are expressed as a fusion protein.

[000121] Many well-known bacterial promoters, including the T7 promoter system, the lactose promoter system, typtophan (Trp) promoter system, Trc/Tac Promoter Systems, beta-lactamase promoter system, tetA Promoter systems, arabinose regulated promoter system, Phage T5 Promoter, or a promoter system from phage lambda, may be employed, and others, as well, and comprise embodiments of the present invention. The promoters will typically control expression, optionally with an operator sequence and may include ribosome binding site sequences for example, for initiating and completing transcription and translation. According to additional embodiments, the vector may also contain expression control sequences, enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter and other necessary information processing sites, such as RNA splice sites, polyadenylation sites and transcription termination sequences as well as any other sequence which may facilitate the expression of the inserted nucleic acid.

[000122] In another embodiment, the present invention comprises methods of use of a polynucleotide, vector, polypeptide and/or fragment thereof as herein described and/or compositions comprising the same in treating, ameliorating, inhibiting or preventing. [000123] The invention also provides transformed cells and progeny thereof into which a nucleic acid molecule encoding humanized antibodies, grafted humanized heavy chains, human light chains or parental CDRs has been introduced by means of recombinant DNA techniques in vitro, ex vivo or in vivo. The transformed cells can be propagated and the introduced nucleic acid transcribed, or encoded protein expressed. It is understood that a progeny cell may not be identical to the parental cell, since there may be mutations that occur during replication. Transformed cells include but are not limited to prokaryotic and eukaryotic cells such as bacteria, fungi, plant, insect, and animal (e.g., mammalian, including human) cells. The cells may be present in culture, in a cell, tissue or organ ex vivo or present in a subject.

[000124] In one embodiment, the term "transformed" means a genetic change in a cell following incorporation of nucleic acid (e.g., a transgene) exogenous to the cell. Thus, a "transformed cell" is a cell into which, or a progeny of which a nucleic acid molecule has been introduced by means of recombinant DNA techniques. Cell transformation to produce host cells may be carried out as described herein or using techniques known in the art. Accordingly, methods of producing cells containing the nucleic acids and cells expressing the humanized antibodies of the invention are also provided.

[000125] Typically cell transformation employs a vector. The term "vector," refers to, e.g., a plasmid, virus, such as a viral vector, or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid, for genetic manipulation (i.e., "cloning vectors"), or can be used to transcribe or translate the inserted polynucleotide (i.e., "expression vectors"). Such vectors are useful for introducing nucleic acids, including a nucleic acid that encodes a humanized antibody operably linked with an expression control element, and expressing the encoded protein in vitro (e.g., in solution or in solid phase), in cells or in vivo.

[000126] A vector generally contains at least an origin of replication for propagation in a cell. Control elements, including expression control elements as set forth herein, present within a vector, are included to facilitate transcription and translation. The term "expression control element" is intended to include, at a minimum, one or more components whose presence can influence expression, and can include components other than or in addition to promoters or enhancers, for example, leader sequences and fusion partner sequences, internal ribosome binding sites (IRES) elements for the creation of multigene, or polycistronic, messages, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA, polyadenylation signal to provide proper polyadenylation of the transcript of a gene of interest, stop codons, etc.

[000127] Vectors can include a selection marker. As is known in the art, "selection marker" means a gene that allows for the selection of cells containing the gene. "Positive selection" refers to a process whereby only cells that contain the selection marker will survive upon exposure to the positive selection. Drug resistance is one example of a positive selection marker; cells containing the marker will survive in culture medium containing the selection drug, and cells which do not contain the marker will die. Such markers include drug resistance genes such as neo, which confers resistance to G418, hygr, which confers resistance to hygromycin, or puro which confers resistance to puromycin, among others. Other positive selection marker genes include genes that allow identification or screening of cells containing the marker. These genes include genes for fluorescent proteins (GFP), the lacZ gene, the alkaline phosphatase gene, and surface markers such as CD8, among others.

[000128] Vectors can contain negative selection markers. "Negative selection" refers to a process whereby cells containing a negative selection marker are killed upon exposure to an appropriate negative selection agent. For example, cells which contain the herpes simplex virus-thymidine kinase (HSV-tk) gene (Wigler et al., Cell 11:223 (1977)) are sensitive to the drug gancyclovir (GANC). Similarly, the gpt gene renders cells sensitive to 6-thioxanthine.

[000129] Mammalian expression systems further include vectors specifically designed for in vivo and ex vivo expression. Such systems include adeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090). AAV vectors have previously been shown to provide expression of Factor IX in humans and in mice at levels sufficient for therapeutic benefit (Kay et al., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)). Adenoviral vectors (U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,928,944), herpes simplex virus vectors (U.S. Pat. No. 5,501,979) and retroviral (e.g., lentivirus vectors are useful for infecting dividing as well as non- dividing cells and foamy viruses) vectors (U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516 and 5,674,703 and WIPO publications W092/05266 and W092/14829) and papilloma virus vectors (e.g., human and bovine papilloma virus) have all been employed in gene therapy (U.S. Pat. No. 5,719,054). Vectors also include cytomegalovirus (CMV) based vectors (U.S. Pat. No. 5,561,063). Vectors that efficiently deliver genes to cells of the intestinal tract have been developed and also may be used (see, e.g., U.S. Pat. Nos. 5,821,235, 5,786,340 and 6,110,456). In yeast, vectors that facilitate integration of foreign nucleic acid sequences into a chromosome, via homologous recombination, for example, are known in the art and can be used. Yeast artificial chromosomes (YAC) are typically used when the inserted nucleic acids are too large for more conventional vectors (e.g., greater than about 12 kb).

[000130] In one embodiment, phagemid vectors for use in the invention include any available in the art suitable for the production of the VH or VL chains/antibodies/antibody libraries/ VL libraries of the present invention and include phagemid vectors pCB04, pITl, pIT2, CANTAB 6, pComb 3 HS. Filamentous vectors and methods of phagemid construction are described in, for example, U.S. Pat. No. 6,054,312 and US Patent 6,803,230 each incorporated herein by reference. Bacteriophage display systems involving non-filamentous bacteriophage vectors known as cytoplasmic bacteriophage or lytic phage can also be utilized as described in for example, US Pat. No. 5,766, 905, incorporated herein by reference.

[000131] Suitable bacterial expression constructs for use with the present invention include, but are not limited to the pCAL, pUC, pET, pETBlue™ (Novagen), pBAD, pLEX, pTrcHis2, pSE280, pSE380, pSE420 (Invitrogen), pKK223-2 (Clontech), pTrc99A, pKK223-3, pRIT2T, pMC1871, pEZZ 18 (Pharmacia), pBluescript II SK (Stratagene), pALTER-Exl, pALTER-Ex2, pGEMEX (Promega), pFivE (MBI), pQE (Qiagen) commercially available expression constructs, and their derivatives, and others known in the art. In another embodiment of the present invention the construct may also include, a virus, a plasmid, a bacmid, a phagemid, a cosmid, or a bacteriophage.

[000132] A humanized antibody of the invention can be generated, identified and isolated from a humanized antibody library, as described and exemplified herein or from other methods of generating humanized antibody libraries that exist in the art which can be used along with a target antigen, to thereby isolate antibody library members that bind specifically to the target antigen. For e.g. kits for generating and screening phage display libraries are commercially available (e.g., the Amersham Biosciences-GE Healthcare Recombinant Phage Antibody System (RPAS), RPAS. Mouse ScFv Module Catalog No. 27-9400-01. In various embodiments, the phage display library is a scFv library or a Fab library. The phage display technique for screening recombinant antibody libraries has been described extensively in the art. Examples of methods and compounds particularly amenable for use in generating and screening antibody display library can be found in, for example, McCafferty et al. International Publication No. WO 92/01047, U.S. Pat. No. 5,969,108 and EP 589,877 (describing, in particular, display of scFv), Ladner et al., U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500 and EP 436,597 (describing, for example, pill fusion); Dower et al., International Publication No. WO 91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527,839 (describing, in particular, display of Fab); Winter et al., International Publication WO 92/20791 and EP 368,684 (describing, in particular, cloning of immunoglobulin variable domain sequences); Griffiths et al., U.S. Pat. No. 5,885,793 and EP 589,877 (describing, in particular, isolation of human antibodies to human antigens using recombinant libraries); Garrard et al., International Publication No. WO 92/09690 (describing, in particular, phage expression techniques); Knappik et al. International Publication No. WO 97/08320 (describing the human recombinant antibody library HuCaI); Salfeld et al., International Publication No. WO 97/29131, describing the preparation of a recombinant human antibody to a human antigen (human tumor necrosis factor alpha), as well as in vitro affinity maturation of the recombinant antibody) and Salfeld et al., U.S. Provisional Patent Application No. 60/126,603, also describing the preparation of a recombinant human antibody to a human antigen (human interleukin-12), as well as in vitro affinity maturation of the recombinant antibody).

[000133] In another embodiment, the term "affinity maturation" refers to the process of enhancing the affinity of an antibody for its antigen. Methods for affinity maturation include but are not limited to computational screening methods and experimental methods. By "computational screening method" herein is meant any method for designing one or more mutations in a protein, wherein said method utilizes a computer to evaluate the energies of the interactions of potential amino acid side chain substitutions with each other and/or with the rest of the protein.

[000134] The invention further provides, in one embodiment, a method of identifying a humanized antibody optimized for affinity to a known target. In another embodiment, antibodies are humanized with retention of high affinity for the antigen and other favorable biological properties. Alternatively, and In another embodiment, the affinity of the humanized antibody for the antigen is higher than the affinity of the corresponding non-humanized, intact antibody or fragment or portion thereof (e.g. the candidate rodent antibody).

[000135] A variety of specific methods, well known to one of skill in the art, may be employed to introduce antibody CDRs (or random sequences substituting for antibody CDRs) into antibody frameworks (see, for example, U.S. application Ser. Nos. 09/434,879 and 09/982,464). In another embodiment, overlapping oligos may be used to synthesize an antibody gene, or portion thereof (for example, a gene encoding a humanized antibody). In another embodiment, mutagenesis of an antibody template may be carried out using the methods of Kunkel (infra), for example to introduce a modified CDR or a random sequence to substitute for a CDR.

[000136] In another embodiment, antibody fragments may be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can, in another embodiment, be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') ₂ - This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R., Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[000137] Antibodies provided herein may be screened using a variety of methods, including but not limited to those that use in vitro assays, in vivo and cell-based assays, and selection technologies. Properties of antibodies that may be screened include but are not limited to stability, solubility, and affinity for the target. Multiple properties may be screened simultaneously or individually. Proteins may be purified or unpurified, depending on the requirements of the assay. In one embodiment, the screen is a qualitative or quantitative binding assay for binding of antibodies to a protein or nonprotein molecule that is known or thought to bind the antibody. In one embodiment, the screen is a binding assay for measuring binding to the target antigen. Automation and high-throughput screening technologies may be utilized in the screening procedures. Screening may employ the use of a fusion partner or label. Binding assays can be carried out using a variety of methods known in the art, including but not limited to FRET (Fluorescence Resonance Energy Transfer) and BRET (Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™ (Amplified Luminescent Proximity Homogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmon Resonance, also known as Biacore), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis, and chromatography including gel filtration. These and other methods may take advantage of some fusion partner or label of the antibody. Assays may employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.

[000138] In another embodiment, the screening of populations of polypeptides such as the altered variable region populations produced by the methods of the invention, involve immobilization of the populations of altered variable regions to filters or other solid substrate. This is particularly advantageous because large numbers of different species can be efficiently screened for antigen binding. Such filter lifts will allow for the identification of altered variable regions that exhibit substantially the same or greater binding affinity compared to the donor variable region. Alternatively, if the populations of altered variable regions are expressed on the surface of a cell or bacteriophage, for example, panning on immobilized antigen can be used to efficiently screen for the relative binding affinity of species within the population and for those which exhibit substantially the same or greater binding affinity than the donor CDR variable region.

[000139] Another affinity method for screening populations of altered variable regions polypeptides is a capture lift assay that is useful for identifying a binding molecule having selective affinity for a ligand (Watkins et. al., (1997)). This method employs the selective immobilization of altered variable regions to a solid support and then screening of the selectively immobilized altered variable regions for selective binding interactions against the cognate antigen or binding partner. Selective immobilization functions to increase the sensitivity of the binding interaction being measured since initial immobilization of a population of altered variable regions onto a solid support reduces non-specific binding interactions with irrelevant molecules or contaminants which can be present in the reaction.

[000140] Another method for screening populations or for measuring the affinity of individual altered variable region polypeptides is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or disassociation between a receptor-ligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (kon) and disassociation rates (koff). Methods for measuring the affinity, including association and disassociation rates using surface plasmon resonance are well known in the arts and can be found described in, for example, Jonsson and Malmquist, Advances in Biosnsors, 2:291-336 (1992) and Wu et al. Proc. Natl. Acad. Sci. USA, 95:6037-6042 (1998). Moreover, one apparatus well known in the art for measuring binding interactions is a BIAcore 2000 instrument which is commercially available through Pharmacia Biosensor, (Uppsala, Sweden).

[000141] In one embodiment, the methods described herein are used to remove antibodies which do not exhibit the desired affinity from the library, to arrive at the "optimized" libraries of the invention, or assemble the antibodies based only on the desired characteristics using molecular biology techniques available in the art and as described herein. [000142] In one embodiment, the term "labeled" refers to antibodies of the invention having one or more elements, isotopes, or chemical compounds attached to enable the detection in a screen. In general, labels fall into three classes: a) immune labels, which may be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopic labels, which may be radioactive or heavy isotopes, and c) small molecule labels, which may include fluorescent and calorimetric dyes, or molecules such as biotin that enable other labeling methods. Labels may be incorporated into the compound at any position and may be incorporated in vitro or in vivo during protein expression.

[000143] Detection methods for identification of binding species within the population of altered variable regions can be direct or indirect and can include, for example, the measurement of light emission, radioisotopes, calorimetric dyes and fluorochromes. Direct detection includes methods that operate without intermediates or secondary measuring procedures to assess the amount of bound antigen or ligand. Such methods generally employ ligands that are themselves labeled by, for example, radioactive, light emitting or fluorescent moieties. In contrast, indirect detection includes methods that operate through an intermediate or secondary measuring procedure. These methods generally employ molecules that specifically react with the antigen or ligand and can themselves be directly labeled or detected by a secondary reagent. For example, an antibody specific for a ligand can be detected using a secondary antibody capable of interacting with the first antibody specific for the ligand, again using the detection methods described above for direct detection. Indirect methods can additionally employ detection by enzymatic labels. Moreover, for the specific example of screening for catalytic antibodies, the disappearance of a substrate or the appearance of a product can be used as an indirect measure of binding affinity or catalytic activity.

[000144] The biophysical properties of antibodies, for example stability and solubility, may be screened using a variety of methods known in the art. Protein stability may be determined by measuring the thermodynamic equilibrium between folded and unfolded states. For example, antibodies of the present invention may be unfolded using chemical denaturant, heat, or pH, and this transition may be monitored using methods including but not limited to circular dichroism spectroscopy, fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As will be appreciated by those skilled in the art, the kinetic parameters of the folding and unfolding transitions may also be monitored using these and other techniques. The solubility and overall structural integrity of an antibody may be quantitatively or qualitatively determined using a wide range of methods that are known in the art. Methods which may find use in the present invention for characterizing the biophysical properties of antibodies include gel electrophoresis, isoelectric focusing, capillary electrophoresis, chromatography such as size exclusion chromatography, ion-exchange chromatography, and reversed-phase high performance liquid chromatography, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorbance spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical ultra-centrifugation, dynamic light scattering, proteolysis, and cross-linking, turbidity measurement, filter retardation assays, immunological assays, fluorescent dye binding assays, protein- staining assays, microscopy, and detection of aggregates via ELISA or other binding assay. Structural analysis employing X-ray crystallographic techniques and NMR spectroscopy may also find use. In one embodiment, stability and/or solubility may be measured by determining the amount of protein solution after some defined period of time. In this assay, the protein may or may not be exposed to some extreme condition, for example elevated temperature, low pH, or the presence of denaturant. Because function typically requires a stable, soluble, and/or well-folded/structured protein, the aforementioned functional and binding assays also provide ways to perform such a measurement. For example, a solution comprising an antibody could be assayed for its ability to bind target antigen, then exposed to elevated temperature for one or more defined periods of time, then assayed for antigen binding again. Because unfolded and aggregated protein is not expected to be capable of binding antigen, the amount of activity remaining provides a measure of the antibody' s stability and solubility.

[000145] The biological properties of the antibodies of the present invention may be further characterized in cell, tissue, and whole organism experiments. As is known in the art, drugs are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure a drug's efficacy for treatment against a disease or disease model, or to measure a drug's pharmacokinetics, toxicity, and other properties. Said animals may be referred to as disease models. With respect to the antibodies of the present invention, a particular challenge arises when using animal models to evaluate the potential for inhuman efficacy of candidate polypeptides-this is due, at least in part, to the fact that antibodies that have a specific effect on the affinity for a human Fc receptor may not have a similar affinity effect with the orthologous animal receptor. These problems can be further exacerbated by the inevitable ambiguities associated with correct assignment of true orthologs (Mechetina et al., Immunogenetics, 2002 54:463-468, incorporated entirely by reference), and the fact that some orthologs simply do not exist in the animal (e.g. humans possess an Fc-γRIIa whereas mice do not). Therapeutics are often tested in mice, including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice (including knockins and knockouts). For example, an antibody of the present invention that is intended as an anti-cancer therapeutic may be tested in a mouse cancer model, for example a xenograft mouse. In this method, a tumor or tumor cell line is grafted onto or injected into a mouse, and subsequently the mouse is treated with the therapeutic to determine the ability of the antibody to reduce or inhibit cancer growth and metastasis. An alternative approach is the use of a SCID murine model in which immune -deficient mice are injected with human Periferal Blood Lymphocytes (PBLs), conferring a semi-functional and human immune system—with an appropriate array of human FcRs- to the mice that have subsequently been injected with antibodies or Fc -polypeptides that target injected human tumor cells. In such a model, the Fc -polypeptides that target the desired antigen (such as her2/neu on SkOV3 ovarian cancer cells) interact with human PBLs within the mice to engage tumoricidal effector functions. Such experimentation may provide meaningful data for determination of the potential of said antibody to be used as a therapeutic. Any organism, e.g., mammals, may be used for testing. For example because of their genetic similarity to humans, monkeys can be suitable therapeutic models, and thus may be used to test the efficacy, toxicity, pharmacokinetics, or other property of the antibodies of the present invention. Tests of the antibodies of the present invention in humans are ultimately required for approval as drugs, and thus of course these experiments are contemplated. Thus the antibodies of the present invention may be tested in humans to determine their therapeutic efficacy, toxicity, pharmacokinetics, and/or other clinical properties.

[000146] In one embodiment, the term "functional fragment" refers to a fragment that maintains a certain degree of biological activity as compared to the wild type despite it being a modified version of the native or wild type antibody or polypeptide. This degree of activity could range from moderate to high as compared to the wild type, where the "activity" refers to its natural biophysical or biochemical characteristics, e.g. binding ability, affinity, half-life, etc.

[000147] The antibodies/polypeptides of the present invention may find use in a wide range of products. In one embodiment the antibody of the invention is a therapeutic, a diagnostic, or a research reagent. In one embodiment, an antibody of the invention is a therapeutic. In another embodiment, the antibody of the present invention may be used for agricultural or industrial uses. An antibody of the present invention may find use in an antibody composition that is monoclonal or polyclonal. The antibodies of the present invention may be agonists, antagonists, neutralizing, inhibitory, or stimulatory. In one embodiment, the antibodies of the present invention are used to kill target cells that bear the target antigen, for example cancer cells. In an alternate embodiment, the antibodies of the present invention are used to block, antagonize, or agonize the target antigen. In an alternate embodiment, the antibodies of the present invention are used to block, antagonize, or agonize the target antigen and kill the target cells that bear the target antigen. [000148] In one embodiment, provided herein is a kit for preparing a library of humanized antibodies, said kit comprising in another embodiment, a library of polynucleotides encoding a human light chain sequence, a polynucleotide encoding a human heavy chain sequence that is similar to the sequence of a murine antibody whose grafting of its CDR is desired, and, a polynucleotide encoding a murine antibody fragment comprising a complementarity determining region (CDR) region, whose grafting onto said human heavy chain similar sequence is desired and reagents for grafting said nucleic acid encoding a murine antibody fragment comprising complementarity determining region (CDR) regions onto said human heavy chain similar sequence. In another embodiment, the kit comprises a vector comprising the polynucleotides encoding human antibody templates. In another embodiment, the kit comprises bacteriophages comprising said polynucleotides encoding human antibody templates.

[000149] In one embodiment, the kit comprises a vector comprising the polynucleotides encoding human VH chains, polypeptides, parental CDRs, VL library, or humanized antibody of the invention, or In another embodiment, the kit comprises bacteriophages comprising the polynucleotides encoding human VH chains, polypeptides, parental CDRs, VL library, or humanized antibody of the invention.

[000150] The invention further provides kits comprising one or more compositions of the invention, including pharmaceutical formulations, packaged into suitable packaging material. In one embodiment, a kit includes a human VH chains, polypeptides, parental CDRs, VL library, or humanized antibody. In another embodiment, a kit includes a nucleic acid encoding human VH chains, polypeptides, parental CDRs, VL library, or humanized antibody of the invention. In additional embodiments, a kit includes nucleic acids that further include an expression control element; an expression vector; a viral expression vector; an adeno- associated virus expression vector; an adenoviral expression vector; and a retroviral expression vector. In yet an additional embodiment, a kit includes a cell that expresses a humanized antibody, antibody template, FR library, or parental CDRs.

[000151] In additional embodiments, a kit includes a label or packaging insert including instructions for expressing a humanized antibody or a nucleic acid encoding a human VH chains, polypeptides, parental CDRs, VL library, or humanized antibody in cells in vitro, in vivo, or ex vivo. In yet additional embodiments, a kit includes a label or packaging insert including instructions for treating a subject (e.g., a subject having or at risk of having asthma) with a humanized antibody or a nucleic acid encoding a humanized antibody, antibody template, FR library, or parental CDRs in vivo, or ex vivo. [000152] In one embodiment, the term "packaging material" refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.). The label or packaging insert can include appropriate written instructions, for example, practicing a method of the invention, e.g., treating the common cold. Kits of the invention therefore can additionally include instructions for using the kit components in a method of the invention.

[000153] Instructions can include instructions for practicing any of the methods of the invention described herein. Thus, invention pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration to a subject. Instructions may additionally include indications of a satisfactory clinical endpoint or any adverse symptoms that may occur, or additional information required by the Food and Drug Administration for use on a human subject.

[000154] In another embodiment, "symptoms" may be any manifestation of a disease or pathological condition as described hereinabove.

[000155] The instructions may be on "printed matter," e.g., on paper or cardboard within the kit, on a label affixed to the kit or packaging material, or attached to a vial or tube containing a component of the kit. Instructions may comprise voice or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as magnetic/optical storage media.

[000156] In another embodiment, the kits of the invention comprise only the backbone and the software to design the antibody templates, or FR libraries that satisfy the scores of the present invention.

[000157] Invention kits can additionally include a buffering agent, a preservative, or a protein/nucleic acid stabilizing agent. The kit can also include control components for assaying for activity, e.g., a control sample or a standard. Each component of the kit can be enclosed within an individual container or in a mixture and all of the various containers can be within single or multiple packages. For example, an invention composition can be packaged into a hand pump container or pressurized (e.g., aerosol) container for spraying the composition into the throat or nasal or sinus passages of a subject. [000158] In one embodiment the compositions of this invention comprise a polypeptide/antibody isolated from the process described herein, alone or in another embodiment, in combination with a second pharmaceutically active or therapeutic agent. In one embodiment, the term "pharmaceutically active agent" refers to any medicament which satisfies the indicated purpose. In another embodiment, the term "agent" of this invention is a decongestant, antibiotic, bronchodilator, anti-inflammatory steroid, leukotriene antagonist or histamine receptor antagonist, and the like.

[000159] In one embodiment, the route of administration may be parenteral, or a combination thereof. In another embodiment, the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation (aerosol), nasal aspiration (spray), intranasal (drops), sublingual, oral, aerosol or suppository or a combination thereof. In one embodiment, the dosage regimen will be determined by skilled clinicians, based on factors such as exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, body weight, and response of the individual patient.

[000160] For intranasal administration or application by inhalation, solutions or suspensions of the compounds mixed and aerosolized or nebulized in the presence of the appropriate carrier suitable. Such an aerosol may comprise any agent described herein.

[000161] For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas. Ampoules are convenient unit dosages. Such a suppository may comprise any agent described herein.

[000162] Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. Such compositions may be formulated for immediate or slow release. It is also possible to freeze-dry the new compounds and use the lyophilisates obtained, for example, for the preparation of products for injection.

[000163] For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[000164] In one embodiment, a composition of or used in the methods of this invention may be administered alone or within a composition. In another embodiment, compositions of this invention admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application which do not deleteriously react with the active compounds may be used. In one embodiment, suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. In another embodiment, the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. In another embodiment, they can also be combined where desired with other active agents, e.g., vitamins.

[000165] Pharmaceutical compositions include "pharmaceutically acceptable" and "physiologically acceptable" carriers, diluents or excipients. In one embodiment, the terms "pharmaceutically acceptable" and "physiologically acceptable" refers to any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of at least one compound for use in the present invention. This term refers to the use of buffered formulations as well, wherein the pH is maintained at a particular desired value, ranging from pH 4.0 to pH 9.0, in accordance with the stability of the compounds and route of administration. The terms include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration. Such formulations can be contained in a liquid; emulsion, suspension, syrup or elixir, or solid form; tablet (coated or uncoated), capsule (hard or soft), powder, granule, crystal, or microbead. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. [000166] Pharmaceutical compositions can be formulated to be compatible with a particular local or systemic route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by particular routes. Specific non-limiting examples of routes of administration for compositions of the invention are inhalation or intranasal delivery. Additional routes include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.

[000167] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

[000168] Pharmaceutical compositions for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can be included in the composition. Including an agent which delays absorption, for example, aluminum monostearate and gelatin can prolong absorption of injectable compositions.

[000169] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of above ingredients followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients as above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[000170] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[000171] The present invention's humanized antibodies/ polypeptides, including subsequences and modified forms and nucleic acids encoding them, can be prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate. The compositions can also be delivered using implants and microencapsulated delivery systems to achieve local or systemic sustained delivery or controlled release.

[000172] Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells or tissues using antibodies or viral coat proteins) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[000173] In one embodiment, polypeptides/antibodies of the present invention are administered as part of a vaccine. In another embodiment, the term vaccine is to be understood to encompass any immunomodulating composition, and such vaccines may comprise an adjuvant, an antigen, an immuno-modulatory compound, or a combination thereof, in addition to the polypeptides of this invention.

[000174] In another embodiment, the adjuvant may include, but is not limited to: (A) aluminium compounds (e.g. aluminium hydroxide, aluminium phosphate, aluminium hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate, etc. [e.g. see chapters 8 & 9 of ref. 96]), or mixtures of different aluminium compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred; (B) MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs, which may be devoid of additional detergent; (E) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic -block polymer L121, and thr- MDP, either micro fluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; (F) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (G) saponin adjuvants, such as QuilA or QS21, also known as Stimulon™; (H) chitosan; (I) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colony stimulating factor, tumor necrosis factor, etc.; (K) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL)]; (L) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions; (M) oligonucleotides comprising CpG motifs] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (N) a polyoxyethylene ether or a polyoxyethylene ester; (O) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol; (P) an immuno-stimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin; (Q) an immuno-stimulant and a particle of metal salt; (R) a saponin and an oil-in-water emulsion; (S) a saponin (e.g. QS21)+3dMPL+IL12 (optionally+a sterol) ; (T) E. coli heat-labile enterotoxin ("LT"), or detoxified mutants thereof, such as the K63 or R72 mutants; (U) cholera toxin ("CT"), or diphtheria toxin ("DT") or detoxified mutants of either ; (V) double- stranded RNA; (W) monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529]; (X) polyphosphazene (PCPP); or (Y) a bioadhesive such as esterified hyaluronic acid microspheres or a mucoadhesive such as crosslinked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose.

[000175] In another embodiment, administration of the compounds of this invention is intended to reduce the severity of the pathologic condition. By the term "reduce the severity of the pathologic condition", it is to be understood that any reduction via the methods, compounds and compositions disclosed herein, is to be considered encompassed by the invention. Reduction in severity may, in one embodiment comprise enhancement of survival, or in another embodiment, halting disease progression, or in another embodiment, delay in disease progression. [000176] In one embodiment, dosing is dependent on the cellular responsiveness to the administered molecules/compounds or compositions comprising same. In general, the doses utilized for the above described purposes will vary, but will be in an effective amount to exert the desired effect, as determined by a clinician of skill in the art. As used herein, the term "pharmaceutically effective amount" refers to an amount of a compound as described herein, which will produce the desired alleviation in symptoms or other desired phenotype in a patient.

[000177] In one embodiment of the invention, the concentrations of the compounds will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration and the individual tolerability of the compositions.

[000178] In another embodiment, any of the compositions of this invention will comprise a compound, in any form or embodiment as described herein. In another embodiment, any of the compositions of this invention will consist of a compound, in any form or embodiment as described herein. In another embodiment, any of the compositions of this invention will consist essentially of a compound, in any form or embodiment as described herein. In another embodiment, the term "comprise" refers to the inclusion of the indicated active agent, such as the compound of this invention, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry.

[000179] It will be appreciated that the actual amounts of active compound in a specific case will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application, and the particular conditions and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.

[000180] In one embodiment, the compounds of the invention may be administered acutely for acute treatment of temporary conditions, or may be administered chronically, especially in the case of progressive, recurrent, or degenerative disease. In one embodiment, one or more compounds of the invention may be administered simultaneously, or in another embodiment, they may be administered in a staggered fashion. In one embodiment, the staggered fashion may be dictated by the stage or phase of the disease.

[000181] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[000182] In another embodiment, the compositions of this invention may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[000183] Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[000184] Furthermore, in another embodiment, the pharmaceutical compositions of this invention are administered as a suppository, for example a rectal suppository or a urethral suppository. Further, in another embodiment, the pharmaceutical compositions are administered by subcutaneous implantation of a pellet. In a further embodiment, the pellet provides for controlled release of an agent over a period of time. In yet another embodiment, the pharmaceutical compositions are administered in the form of a capsule.

[000185] In one embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the anti-estrogen compound is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate -release composition, i.e. a composition in which the entire compound is released immediately after administration. In one embodiment, the controlled- or sustained-release compositions of the invention are administered as a single dose. In another embodiment, compositions of the invention are administered as multiple doses, over a varying time period of minutes, hours, days, weeks, months or more. In another embodiment, compositions of the invention are administered during periods of acute disease. In another embodiment, compositions of the invention are administered during periods of chronic disease. In another embodiment, compositions of the invention are administered during periods of remission. In another embodiment, compositions of the invention are administered prior to development of gross symptoms.

[000186] In yet another embodiment, the pharmaceutical composition of this invention is delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump is used. In another embodiment, polymeric materials are used. In yet another embodiment, a controlled release system is placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose. In another embodiment, the controlled-release system is a controlled release system known in the art.

[000187] In one embodiment, the compositions also include incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[000188] In one embodiment, the preparation of pharmaceutical compositions that contain an active component, for example by mixing, granulating, or tablet-forming processes, is well understood in the art. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the compound is mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. For parenteral administration, the compound is converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other substances.

[000189] In another embodiment, an active component is formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[000190] For use in medicine, the salts are pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts, which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methane sulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[000191] In another embodiment, the antibodies and antibody portions of the present invention are administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection, intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.

[000192] In certain embodiments, an antibody or antibody portion of the invention are orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[000193] Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents.

[000194] Depending on the type of antibody desired, various animal hosts may be used for in vivo immunization. A host that itself expresses an endogenous version of the antigen(s) of interest can be used or, alternatively, a host can be used that has been rendered deficient in an endogenous version of the antigen(s) of interest. For example, it has been shown that mice rendered deficient for a particular endogenous protein via homologous recombination at the corresponding endogenous gene (i.e., "knockout" mice) elicit a humoral response to the protein when immunized with it and thus can be used for the production of high affinity monoclonal antibodies to the protein. (See, e.g., Roes et al. (1995) Journal of Immunological Methods 183:231-237; Lunn et al. (2000) Journal of Neurochemistry 75:404-412). [000195] In one embodiment, the term "position" refers to a location in the sequence of a protein. Positions are typically, but not always, numbered sequentially. For example, position 297 is a position in the human antibody IgGl. By "residue", in one embodiment, is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (or Asn297 or N297) is a residue in the human antibody IgGl. By "variant protein sequence" In another embodiment, is meant a protein sequence that has one or more residues that differ in amino acid identity from another similar protein sequence. Said similar protein sequence may be the natural wild type protein sequence, or another variant of the wild type sequence.

[000196] In another embodiment, the term "epitope" is defined herein as a region of the antigen that binds to the antibody. In general, epitopes are comprised by local surface structures that can be formed by contiguous or noncontiguous amino acid sequences.

[000197] In another embodiment, the term "immunize" refers herein to the process of presenting an agonistic antigen to an immune repertoire whether that repertoire exists in a natural genetically unaltered organism, or a transgenic organism modified to display an artificial human immune repertoire. Similarly, an "immunogenic preparation" is a formulation of antigen that contains adjuvants or other additives that would enhance the immunogenicity of the antigen. An example of this would be coinjection of a purified form of GLP-I receptor with Freund's complete adjuvant into a mouse. "Hyperimmunization", as defined herein, is the act of serial, multiple presentations of an antigen in an immunogenic preparation to a host animal with the intention of developing a strong immune response.

[000198] Although the pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical composition suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with little, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and other mammals.

[000199] In one embodiment, "preventing, or treating" refers to any one or more of the following: delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics. In one embodiment, "treating" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described hereinabove.

[000200] In one embodiment, the antibodies/polypeptides of the present invention can be tested in a variety of orthotopic tumor models. These clinically relevant animal models are important in the study of pathophysiology and therapy of aggressive cancers like pancreatic, prostate and breast cancer. Immune deprived mice including, but not limited to athymic nude or SCID mice are frequently used in scoring of local and systemic tumor spread from the site of intraorgan (e.g. pancreas, prostate or mammary gland) injection of human tumor cells or fragments of donor patients.

[000201] In embodiments, antibodies of the present invention may be assessed for efficacy in clinically relevant animal models of various human diseases. In many cases, relevant models include various transgenic animals for specific tumor antigens.

[000202] In one embodiment, the testing of antibodies may include study of efficacy in primates (e.g. cynomolgus monkey model) to facilitate the evaluation of depletion of specific target cells harboring target antigen, specifically in therapeutic studies of autoimmune, transplantation, and cancer. Additional primate models include but are not limited to that of the rhesus monkey.

[000203] Toxicity studies are performed to determine the antibody effects that cannot be evaluated in standard pharmacology profile or occur only after repeated administration of the agent. Most toxicity tests are performed in two species-a rodent and a non-rodent-to ensure that any unexpected adverse effects are not overlooked before new therapeutic entities are introduced into man. In general, these models may measure a variety of toxicities including genotoxicity, chronic toxicity, immunogenicity, reproductive/developmental toxicity and carcinogenicity. Included within the aforementioned parameters are standard measurement of food consumption, bodyweight, antibody formation, clinical chemistry, and macro- and microscopic examination of standard organs/tissues (e.g. cardiotoxicity). Additional parameters of measurement are injection site trauma and the measurement of neutralizing antibodies, if any. Traditionally, monoclonal antibody therapeutics, naked or conjugated are evaluated for cross-reactivity with normal tissues, immunogenicity/antibody production, conjugate or linker toxicity and "bystander" toxicity of radiolabeled species. Nonetheless, such studies may have to be individualized to address specific concerns and following the guidance set by ICH S6 (Safety studies for biotechnological products also noted above). As such, the general principles are that the products are sufficiently well characterized and for which impurities/contaminants have been removed, that the test material is comparable throughout development, and GLP compliance.

[000204] The term "operably linked" means that the nucleic acid is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[000205] The term "about" as used herein means in quantitative terms plus or minus 5%, or In another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

[000206] The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects.

[000207] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES Materials and Methods:

Antibodies, antigens, and cell lines

[000208] The humanization method was tested using a mouse antibody, M225. M225 binds specifically to human EGFR. Recombinant EGFR-Fc fusion proteins was purchased from R & D Systems Inc. Human tumor cell lines, DiFi (colorectal carcinoma) and BxPC3 (pancreatic carcinoma) were obtained from ATCC.

Design of CDR grafted VH chains

[000209] The most homologous human VH sequence to that of the original murine antibody were identified by searching against all non-redundant GenBank CDS databases by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). During the search, CDR residues in the KABAT definition were masked and only mouse FR residues were aligned with amino acid sequences of functional human antibodies. AAB67785 (Rassenti and Kipps, 1997) was selected as human VH FRs for M225, because it has high sequence homology (amino acid identity = 67.8%; similarity = 89.7%), and belong to the same subgroup of the original VH (human heavy chain subgroup II).

[000210] Using the AAB67785 sequence, two humanized VH chains were designed, in which two different set of CDRs, as defined Kabat and Chothia definitions (Kabat et al., 1977; Chothia et al, 1989), respectively, were grafted to the human FR (Fig. 1). The CDR-grafted VH chains were then used to create 225Vc and 225Vd libraries, respectively.

Library construction

[000211] In constructing new libraries for humanization, the single grafted VH gene segment was cloned into the large Fab phage display library replacing the original diverse VH repertoire.

[000212] Genes encoding the grafted VH chain were synthesized commercially and subcloned into pUCminusMCS vector (Blue Heron Biotech). To attach CHl gene, the synthesized VH gene was amplified by PCR, and digested with BstEII at the C-terminus. The CHl gene was amplified by PCR from pCESl phagemid vector (de Haard et al., 1999), and digested with BstEII at the N-terminus. Subsequently, the synthesized VH and CHl genes were ligated together, and amplified by PCR again. After digestion with Ncol and Notl, VH-CHl fragment was cloned back to pCESl vector.

[000213] For the source of the VL repertories, a human Fab phage display library vector was used. The library phagemid was first digested with Sfil and Notl, treated with antarctic phosphatase (NEB), and purified by agarose gel electrophoresis to remove VH-CHl gene fragment from the VL chain-containing backbone vector. The grafted VH-CHl fragment described above was amplified by PCR from pCESl vector, digested with Sfil and Notl, and replaced VH gene in library vector. Thus, in new libraries, a single grafted VH was paired with 10 ⁷ -10 ⁸ VL repertories. E. coli TGl cells (Stratagene) were transformed with the new library phagemid by electroporation, and grown on ten large 2YT-AG plates (2YT medium plate containing 2% glucose and 100 μg/ml ampicillin). At the same time, small amount of transformants was used for titration to confirm library size. After incubation overnight at 30 ⁰ C, all colonies on the plates were scraped into 25 ml 2YT- AG medium, mixed with 8 ml 80% glycerol, and stored at -80 ⁰ C as the library stock.

Selection

[000214] The library stock (100 μl, ~2 x 10 ⁹ cells) was grown to log phase in 100 ml 2YT-AG medium. 10 ml of the culture (1.7 x 10 ⁹ cells) was rescued with M13KO7 helper phage (3.5 x 10 ° pfu), and cultured overnight in 50 ml 2YT-AK medium (2YT medium containing 100 μg/ml ampicillin and 50 μg/ml kanamycin) at 30 ⁰ C. The amplified Fab-displaying phages were precipitated in 4% PEG/0.5 M NaCl, and re- suspended in 1 ml PBS. The phage library (containing at least 10 phage representatives) was blocked with 2% milk/PBS for 30 minutes at room temperature.

[000215] Maxisorp Star tubes (Nunc) were coated with various concentrations of EGFR-Fc (R & D Systems) overnight 4 ⁰ C. The tubes were blocked with 2% milk/PBS at 37 ⁰ C for 2 hours and then incubated with the phage at room temperature for 2 hours. The tubes were washed with PBST (PBS containing 0.05% Tween 20) and PBS. The bound phages were eluted at room temperature for 10 min with 1 ml of 100 mM triethylamine (Sigma), and neutralized with 0.5 ml of IM Tris-HCl (pH 7.4). The half of the eluted phages (750 μl) were added to 10 ml of mid-log phase TGl cells and incubated for 30 min at 37°C. The infected TGl cells were then plated onto large 2YT-AG plates and grown overnight at 30 ⁰ C. At the same time, colony forming units of output phages was tested using small amount of infected TGl cells. All of the colonies were scraped into 6ml 2YT- AG medium, and stored at -80 ⁰ C with 16 % glycerol. For subsequent rounds of selection, 50-100 μl of phage stock was grown in 50 ml 2YT- AG medium and Fab displaying phages were prepared as described above.

Phage ELISA

[000216] Maxisorp 96 well microtiter plates were coated with EGFR-Fc, MgG, or anti-human Fab antibody (1 μg/ml x 100 μl/well) and incubated at 4°C overnight, washed three times with PBS and blocked with 2% milk/PBST for 2 hours at 37°C. After several rounds of selection, individual output colonies were randomly picked and grown in 96 well plates, and Fab-displaying phages were rescued as described above. 100 μl of each phage were blocked with 2% milk/PBST for 1 hour at room temperature, transferred to the coated Maxisorp plates, and incubated for 2 hours at room temperature. The plates were washed three times with PBST, and incubated with anti-M13 phage-HRP conjugate for 1 hour at room temperature. After washing the plates three times with PBST, TMB peroxidase substrate (KPL) was added, and the absorbance at 450 nm was read using microplate reader.

DNA Sequence

[000217] The DNA sequences of the clones randomly picked from the library and the selected Fab clones were determined by dideoxynucleotide sequencing. Classification of the VL genes was performed using IMGT/V-QUEST (Brochet et al., 2008). The guide-tree demonstrating the degree of divergence between the selected sequences were calculated by the neighbor-joining method (Saitou and Nei, 1987) using AlignX software (Invitrogen).

Soluble Fab ELISA

[000218] Phagemid of individual selected clone was used to transform a nonsuppressor E. coli HB2151 cells, and the soluble Fab (sFab) protein was expressed and purified from the periplasmic extract. The cells were cultured till mid- log phase in 2YT- A medium (2YT containing 100 μg/ml ampicillin) supplemented with 0.1% glucose and sFab expression was induced by ImM isopropyl-1-thio-β-D-galactopyranoside at 30 ⁰ C for overnight. The harvested cells were resuspended in the extraction buffer (0.2 M NaBCh, 0.16 M NaCl, 1 mM EDTA, pH8.0) followed by incubation on ice for 30 min. After centrifugation at 10,000 rpm for 15 min, the supernatant was dialyzed with the column buffer (20 mM Tris-HCl, 200 mM NaCl). The sFab was purified by 6xHis tag using Talon metal affinity resin (Clonetech). BIAcore analysis

[000219] The binding kinetics of sFab was measured using a BIAcore biosensor (GE healthcare). EGFR-Fc protein was immobilized onto a CMS sensor chip using amine coupling. Association rate k _on , dissociation k _of τ and the affinity constant K _d were evaluated using the BIA Evaluation 2.0 program.

Receptor phosphorylation assay

[000220] Tumor cells BxPC-3 were grown for 4 hours in 96-well plates, and then cultured overnight in serum free medium till 95% confluent. The cells were incubated with various amounts of antibodies at RT for 30 minutes, followed by stimulation with EGF (8 ng/ml) for an additional 15 minutes. The cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM PMSF, 0.5 mM Na ₃ VO _4, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin), the lysates were transferred to a 96 well plate coated with a mouse monoclonal anti-human EGFR antibody (BD Pharmingen Inc.) and incubated at RT for 60 minutes. The amount of EGFR phosphorylation was determined by the addition of a HRP- conjugated mouse monoclonal anti-phosphotyrosine antibody (Santa Cruz Biotechnology Inc). The total amount of the receptor protein in each well was determined using a rabbit polyclonal anti-human EGFR antibody (Santa Cruz Biotechnology Inc) followed by HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology Inc.). The plates were washed 5 times, TMB peroxidase substrate added, and the absorbance at 450 nm read using a microplate reader (Molecular Device).

Cell proliferation assay

[000221] 1.5 x 10 ⁴ DiFi cells in 100 μl of complete medium were seeded in each well of 96-well plates and cultured for 4 hours. Various amounts of the antibodies were added to wells and allowed to culture for 3 days, after which 10 μl of 5 mg/ml protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated for an additional 3 h. The plates were washed twice with PBS and incubated with 100 μl of isopropanol at room temperature for 15 min, followed by optical density reading at 570 nm.

Construction of Vc/Vd chimeric VH chain

[000222] Four oligonucleotide primers were commercially synthesized (Invitrogen), which encode both strands of the center region of FR2 and CDR2 where the sequences are common in the VH genes of Vc and Vd. The N-terminus half VH genes including FR1-(1/2)FR2 or FR1-(1/2)CDR2, and the C-terminus half VH genes including (1/2)FR2-FR4 or (1/2)CDR2-FR4 were amplified by PCR. Subsequently, the chimeric VH genes were amplified by assembly PCR of N-terminus Vc fragment and C-terminus Vd fragment, or vice versa, using overlapping regions, and cloned back into the phagemid vector paired with the VL chain of the clone Vc-2A6 which were selected from Vc library for EGFR binding described above.

EXAMPLE 1: LIBRARY CONSTRUCTION

[000223] For proof-of-concept of new "grafting and shuffling" humanization strategy, two libraries for C225 (225Vc-SEQ ID NO: 2 and 225Vd-SEQ ID NO: 3) were constructed. Those libraries contained large human VL chain library paired with the single VH chain in which two different set of CDRs were grafted, that is, Kabat CDRs in Vc and Chothia CDRs in Vd (Fig. 1). The size of 225Vc and 225Vd libraries was estimated as 4.6 x 10 ^s and 1.8 x 10 ⁸ , respectively, by colony-forming units after electroporation into TGl cells. Agarose gel electrophoresis and sequence analysis of 40 randomly picked clones revealed about 95% of the library contains full length Fab, in which the designed VH chain was combined with all different VL chain, indicating a large diversity of the libraries.

EXAMPLE 2: SELECTION OF ANTI-EGFR AND ANTI-KDR CLONES

[000224] To test efficiency of humanization, 225Vc (SEQ ID NO: 2) and 225Vd (SEQ ID NO: 3) libraries were used for selection against EGFR by phage display. After each round selection, recovered phage clones were picked randomly and isolated phages were subjected to phage ELISA for EGFR binding activity. Humanized antibody clones were successfully isolated from 225Vc library. After the first and the second rounds of selection, 5 out of 80 clones (6%) and 86 out of 92 clones (93%) recovered were confirmed as specific EGFR binders, respectively.

[000225] Table 1. Binding kinetics of the humanized Fabs to human EGFR k _on (x 10 ⁵ M- ¹ S ^"1 ) k _off (xlO ^"4 s ^"1 ) K _D (xlO ⁹ M)

M225 9.0 8.5 0.9

2A4 6.8 53.0 7.8

2A6 8.1 15.0 1.9

2A10 9.9 49.0 5.0

2C5 n.d. n.d. n.d.

2C8 7.5 61.0 8.1

2Cl 1 3.4 49.0 15.0

2El 7.5 61.0 8.1

2Fl 1 n.d. n.d. n.d.

2G12 n.d. n.d. n.d.

Affinity measurements were carried out by BIAcore.

[000226] From 225Vd library, no positive clone was obtained even after 3 rounds of selection. Further round of selection was not performed, because phage ELISA showed that after 3 rounds of selection, the phages with an affinity to Fc fragment or no-Fab displaying phages started to be concentrated in the output fractions. This suggests that grafting of the Chothia defined CDRl and CDR2 for VH chain were not sufficient to retain antigen binding (see below).

[000227] The DNA sequences of the selected 86 clones were analyzed and showed that all clones contained the same VH sequence designed for the 225Vc library, except for one point mutation in Vc-2E6, in which Gly44 were replaced with Ser. In total, 46 new VL sequences were identified among of 86 active clones.

[000228] Figure 2 shows the EGFR binding activities of 46 unique antibodies assayed by phage ELISA (Fig. 2a) and their sequence diversity in the VL chain (Fig. 2b). The clones repeatedly isolated (Vc-2A6; 32 clones, Vc-2E1; 6 clones, Vc-2C8; 4 clones, and Vc-2A10; 2 clones) had higher binding activities. The selected VL sequences were classified using IMGT/V-QUEST (http://imgt.cines.fr/; Brochet et al., 2008) and V-BASE2 (http://www.vbase2.org/vbase2.php; Retter et al., 2005). All VL genes were identified as IGkV3 family, except for Vc-2F5 (IGkVl family). Highly active clones were clustered in the diversity tree and were most homologous to IGkV3-15 germline.

[000229] CDR sequence alignment of 9 most active clones revealed that the selected CDRs (SEQ ID NOs: 17-22) were similar to that of the parental M225 VL, but not totally identical (Fig. 3). EXAMPLE 3: QUANTITATIVE ANALYSIS OF EGFR BINDING BY HUMANIZED ANTIBODIES

[000230] Soluble Fab fragments of several selected clones were produced and their binding efficiencies were compared with that of M225 Fab quantitatively. Consistent with the phage ELISA (Fig. 2a), the result showed that the dominant clone Vc-2A6 binds efficiently to EGFR among all other Fabs, and comparable to the parental M225 Fab (Fig. 4a). The affinities of clones Vc-2A4, Vc-2C8, Vc-2C11, and Vc-2E1, which share same CDR sequences (Fig. 3), and Vc-2A10 were slightly weaker than those of Vc-2A6 and M225. Clones Vc-2C5, Vc-2F11 and Vc-2G12 did not show strong binding to EGFR.

[000231] The binding kinetics of various humanized Fabs was determined by BIAcore analysis (Table 1). The K _D value of the best clone Vc-2A6 Fab was 1.9 nM, ~2 fold loss of affinity compared to the parental M225, mainly because of higher k _off value. The humanized Fabs, Vc-2A4, Vc-2A10, Vc-2C8, Vc-2C11, and Vc-2E1, bound to EGFR with similar Ic _0n rates but faster k _off rates than parental Fab, resulted in 5-10 fold weaker affinity. Clone Vc-2C5, Vc-2F11, and Vc-2G12 did not show detectable binding, coincident with the result by ELISA for sFab (Fig. 4a).

EXAMPLE 4: INHIBITION OF EGF-STIMULATED EGFR PHOSPHORYLATION AND CELL

PROLIFERATION

[000232] The biological activity of the humanized antibodies on EGF-induced EGFR phosphorylation and cell proliferation were evaluated on cancer cell lines BxPC3 and DiFi.

[000233] In the phosphorylation assay, the levels of EGFR phosphorylation stimulated by EGF were measured in the presence of various humanized antibodies (Fig. 4b). The results showed that the clone Vc- 2A6 Fab was able to inhibit EGFR phosphorylation on tyrosine residues in intact cells (Fig. 4b), although other Fabs did not have obvious effect.

[000234] In the proliferation assays, the effect of the humanized antibodies on cell growth was examined (Fig. 4c). We first observed that the clone Vc-2A6 Fab inhibited growth of cells to the same level of M225 Fab (Fig. 4c), although the effect of the other Fabs was marginal. EXAMPLE 5: IDENTIFICATION OF IMPORTANT RESIDUES IN VH CHAIN, WHICH SHOULD

BE GRAFTED AS CDR RESIDUES

[000235] Since the FR and CDR3 residues were same in the VH chains of Vc and Vd, only 10 residues around CDRl and CDR2 were different depending on the Kabat and Chothia definitions (Fig. 1). To confirm which residues are important for antigen binding specificity, VH chain of Vd library and various Vc/Vd chimeric VH chains were paired with the selected VL chain of Vc-2A6, and tested for EGFR binding.

[000236] Phage ELISA result showed that Vd-2A6 did not show any binding, but all chimeric VH chains were active (Fig. 5). Compared to Vd which has Chothia CDRl and CDR2, F-CD (Kabat CDRl and Chothia CDR2) and F-DC (Chothia CDRl and Kabat CDR2) binds to EGFR, indicating grafting of one of Kabat defined CDRl and CDR2 retains antigen binding activity. The activity of F-DC was ~30-fold higher than that of F-CD, indicating CDR2 is more important for binding. The results also showed that the part of Kabat CDR2 recovers the activity. For example, gain of mouse type residues DTPFT at the positions H58 and 61-64 (C-DC) recovers the binding to some extend, and further mouse type mutation VaI at H50 (F-DC) improve the activity about 4-fold. Similarly, compared to Vc in which CDRs were both Kabat defined, the loss of mouse residues DTPFT at 58 and 61-64 (C-CD) decrease binding slightly (4-fold), and the further loss of VaI at H50 (F-CD) affect more strikingly (17-fold). The results indicate that the residues at H50 and H58-64 were important for retaining antigen binding, thus Kabat defined CDR2 should be grafted. Also, if compared Vc and F-DC, or F-CD and Vd, the activities of the Kabat CDRl grafted VH chains (Vc and F-CD) bound better than the Chothia CDRl grafted VH chains (F-DC and Vd, respectively), indicating Kabat CDRl definition should be applied. Overall, the results demonstrate that Kabat defined CDRs should be grafted to the human FRs.