WO2010056893A1

WO2010056893A1 - Humanization and affinity-optimization of antibodies

Info

Publication number: WO2010056893A1
Application number: PCT/US2009/064247
Authority: WO
Inventors: Jaafar Nassar Sleiman Haidar; Qing-An Yuan; Zhenping Zhu
Original assignee: Imclone Llc
Priority date: 2008-11-13
Filing date: 2009-11-12
Publication date: 2010-05-20

Abstract

In an efficient selection of higher affinity binders for an antibody humanization strategy, original CDRs are grafted, but no homology modeling is required to determine the key residues in a framework region. One human variable region heavy chain or "VH' and one variable region light chain or 'VL" germline segments show characteristics of high expression in E. coli as a universal template. Key residues in the framework scaffold were determined by screening of available antibody crystal structure data as well as published documents that described the refinements of a specific mouse humanization project. Diversified residues that represent either human antibody selections or mouse selections were integrated into those key framework positions that were assumed to be important in either supporting CDRs or affecting the overall domain structure. A phage display Fab library was used to screen functional reshaped antibodies, which facilitates the isolation of well-maintained clones in a standard panning.

Description

HUMANIZATION AND AFFINITY-OPTIMIZATION OF ANTIBODIES

FIELD OF INVENTION

[001] The present invention relates to affinity-optimized antibodies, libraries comprising the same and methods of production thereof.

BACKGROUND OF THE INVENTION

[002] 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.

[003] 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 m 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

[004] In one embodiment, provided herein is an isolated optimized human antibody comprising a heavy and light chain of a human antibody, whose overall sequence shares a low sequence similarity with that of a murine antibody, wherein a framework (FR) region in said human antibody possesses residues comprising a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score, whereby:

i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) is determined as the ratio between the number of complementarity determining region (CDR) residues within 6A contact of an FR residue and the number of total residues within όA contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%; ii) a positive B to M score for particular residues in a Framework Region (FR) is determined as the frequency of Framework Region (FR) residue mutations which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding; and iii) a positive human diversity score is determined as a frequency of diversity at an Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0 45

[005] hi one embodiment, provided herein is a library of optimized human antibody templates for murine complementarity determining region (CDR) grafting, each antibody template in said library comprising a heavy and light chain of a human antibody, whose overall sequence shares a low sequence similarity with that of a murine antibody, wherein grafting of a complementarity determining region (CDR) of said muπne antibody onto said human antibody is desired, and,

i) an FR region in said human antibody in (a) possessing residues comprising a positive complementarity determining region (CDR) contact ratio score or a positive B to M score and a positive human diversity score, or a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score, whereby. ii) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) is determined as the ratio between the number of complementarity determining region (CDR) residues withm βA (Angstroms) contact of an FR residue and the number of total residues withm 6A contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%; in) a positive B to M score for particular residues in a Framework Region (FR) is determined as the frequency of Framework Region (FR) residue mutations which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding; and iv) a positive human diversity score is determined as a frequency of diversity at an Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0 45

[006] In one embodiment, provided herein is a process for preparing a library of affinity-optimized antibody templates, said process comprising

I) compiling structural and sequence information regarding a large non-redundant dataset of human antibodies of known specificity and assigning: i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity ; or, ii) a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, whereby,

II) said positive contact ratio or positive B to M score is combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; or,

III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of said positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or said positive B to M score and a positive human diversity score

[007] In one embodiment, provided herein is a process for preparing a library of affinity-optimized antibody templates, said process comprising

I) compiling structural and sequence information regarding a large non-redundant dataset of human antibodies of known specificity and assigning: i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity , or, ii) a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; whereby,

II) said positive contact ratio or positive B to M score is combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; wherein said human diversity score is determined as a frequency of diversity at a Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45 ; or, III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of said positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or said positive B to M score and a positive human diversity score

[008] In one embodiment, provided herein is a method of identifying a humanized antibody optimized for affinity to a known target, said method comprising the steps of

I) compiling structural and sequence information regarding a large non redundant dataset of human antibodies of known specificity and assigning i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity,

II) a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, whereby,

II) said positive contact ratio or positive B to M score is further combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity,

III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score,

IV) grafting a complementarity determining region (CDR) of a murine antibody of desired specificity onto each antibody template in said library,

V) determining a respective binding affinity for a target for each of said antibodies in said library formed in, and

VI) identifying an antibody from said library having the highest binding affinity for said target

[009] In one embodiment, provided herein is a method of identifying a humanized antibody optimized for affinity to a known target, said method comprising the steps of

I) compiling structural and sequence information regarding a large non redundant dataset of human antibodies of known specificity and assigning: i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, u) a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity,

II) said positive contact ratio or positive B to M score is further combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, wherein said human diversity score is determined as a frequency of diversity at a Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0 45 , or,

IV) grafting a complementarity determining region (CDR) of a murine antibody of desired specificity onto each antibody template in said library;

V) determining a respective binding affinity for a target for each of said antibodies in said library, and,

[0010] In another aspect of the invention, the contact ratio score is determined as the ratio between the number of complementarity determining region (CDR) residues within όA contact of an Framework Region (FR) residue and the number of total residues within 6A contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25% In another embodiment, the B to M score is determined as the frequency of Framework Region (FR) residue mutation which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding

BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 depicts sequence diversity determined for the selected chains (SEQ ID NOs 1-17) Multiple alignments of the human light and heavy Fv sequences were extracted from the KabatMan Server, then, the ammo acid distributions were extracted for each position (numbered according to the Enhanced Chothia scheme) Shannon entropy was used to compute the diversity of ammo acids at any position of the Fv region [0012] Figure 2 depicts a side-by-side comparison of the CDR contact ratio scores of the FR residues in bound and unbound antibodies showed no significant difference in the FR3 of the heavy (A) and light (B) chains of antibodies.

[0013] Figure 3 demonstrates sequence variability at specific positions when comparing murine to human sequences (The direction of the arrows points towards decreasing frequency of the substitution).

[0014] Figure 4 (A) The CDR Contact Ratio plotted for the light FRl The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets 62 Bound (Black) and 75 Unbound (White) antibody structures Within the two datasets, The FR regions share at most 85% sequence identity (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the light FRl. (C) Demonstrates the light FRl amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The ammo acid distribution at each position is computed from the human Fv sequences culled KabatMan database

[0015] Figure 5 (A) The CDR Contact Ratio plotted for the light FR2 The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets 62 Bound (Black) and 75 Unbound (White) antibody structures Within the two datasets, The FR regions share at most 85% sequence identity (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the light FR2 (C) Demonstrates the light FR2 ammo acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The ammo acid distribution at each position is computed from the human Fv sequences culled KabatMan database

[0016] Figure 6 (A) The CDR Contact Ratio plotted for the light FR3 The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets 62 Bound (Black) and 75 Unbound (White) antibody structures Within the two datasets, The FR regions share at most 85% sequence identity (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the light FR3 (C) Demonstrates the light FR3 amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The ammo acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0017] Figure 7 (A) The CDR Contact Ratio plotted for the light FR4 The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets 62 Bound (Black) and 75 Unbound (White) antibody structures Within the two datasets, The FR regions share at most 85% sequence identity (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the light FR4. (C) Demonstrates the light FR4 amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The amino acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0018] Figure 8 (A) The CDR Contact Ratio plotted for the heavy FRl. The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets' 62 Bound (Black) and 75 Unbound (White) antibody structures. Within the two datasets, The FR regions share at most 85% sequence identity. (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the heavy FRl. (C) Demonstrates the heavy FRl amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The ammo acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0019] Figure 9 (A) The CDR Contact Ratio plotted for the heavy FR2. The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets: 62 Bound (Black) and 75 Unbound (White) antibody structures. Within the two datasets, the FR regions share at most 85% sequence identity. (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the heavy FR2. (C) Demonstrates the heavy FR2 ammo acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The amino acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0020] Figure 10 (A) The CDR Contact Ratio plotted for the heavy FR3 The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets: 62 Bound (Black) and 75 Unbound (White) antibody structures. Within the two datasets, The FR regions share at most 85% sequence identity. (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the heavy FR3 (C) Demonstrates the heavy FR3 amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The amino acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0021] Figure 11 (A) The CDR Contact Ratio plotted for the heavy FR4. The CDR Contact Ratio for each FR position is averaged from two antibody structural datasets: 62 Bound (Black) and 75 Unbound (White) antibody structures. Within the two datasets, The FR regions share at most 85% sequence identity. (B) Demonstrates the count or frequency of Back to Mouse mutations at each position of the heavy FR4. (C) Demonstrates the heavy FR4 amino acid variability defined as the Shannon entropy of the observed human amino acid distribution at each FR position The amino acid distribution at each position is computed from the human Fv sequences culled KabatMan database.

[0022] Figure 13 correlates CDR contact Ratio with the Back to Mouse counts of the light and heavy FR positions. The Spearman correlation coefficient is 0.7 (P-value: 9xlO^~4) for the heavy chain FR positions. The correlation in the light chain FR positions is not significant (r = 0.3; P-value: 0.3). This correlation only includes the FR positions m both the heavy and light chain that have a back to mouse count greater or equal to 4.

[0023] Figure 14 illustrates selection conducted as described in Example 5, resulting in a proposed humanization sequence (SEQ ID NOs: 18-31).

[0024] Figure 15 shows a designed heavy chain template, and a derived series of heavy chain fragments with putative hot spots (SEQ ID NOs: 32-40). Similarly, a light chain template was designed, and a series of fragments with putative hot spots was derived (SEQ ID NOs: 41-46).

[0025] Figure 16 illustrates a derived final library, where the heavy chain diversity is 18342, the light chain diversity is 384, and the total library size is 6 x 10⁶ (SEQ ID NOs: 64-71).

[0026] Figure 17 illustrates the anti-EGFR binding ability of 10 Fab clones, as determined by ELISA, that were derived from the competed panning.

[0027] Figure IS Multi-alignment of phage ELISA screening positive clones in VH (A) and VL (B) chains. For ease of illustration deviations (mutations) from the conserved heavy chain sequence (SEQ ID NOs: 62-63) have been tabulated.

DETAILED DESCRIPTION OF THE INVENTION

[0028] Antibody therapy is a promising alternative for the treatment of a number of different diseases. Despite enormous technological strides forward in antibody-based therapies, the development of an antibody exhibiting a high affinity and specificity for its target with minimal to no rejection by the host, has proven challenging. In this invention, although original CDRs are grafted, no homology modeling is required to determine the key residues in a framework region. In addition, it is not a prerequisite to pick a homologous human antibody template as an acceptor. Instead, one human variable region heavy chain or "VH" and one variable region light chain or "VL" germline segments characteristics of high expression in E. cσli and superior physiological properties were used and it is applied as a universal template. Most importantly, key residues in the framework scaffold were determined by systematically screening and analyzing available antibody crystal structure data as well as published documents that described the refinements of a specific mouse humanization project Amino acid choices that represent the human antibody diversity were integrated into those key framework positions that assumed to be important m either supporting CDR or affecting the overall domain structure. A phage display Fab library was used to screen functional reshaped antibodies, which facilitates the isolation of well- maintained clones in a standard panning. In short, the method described herein is time and cost efficient and allow selection of even higher affinity binders as compared to the wild type mouse antibody.

[0029] In another embodiment, the term "acceptor" is intended to mean an antibody molecule or fragment thereof which is to receive the donated portion from the parent or donor antibody molecule or fragment thereof. An acceptor antibody molecule or fragment thereof is therefore imparted with the structural or 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.

[0030] In one embodiment, a CDR contact consensus exists in antibody framework residues where heavy chain framework residues that contact CDRs tend to be more diverse in amino acid sequence. In another embodiment, this consensus does not vary when comparing bound and unbound states of antibodies. Upon integrating structural, sequential and experimental data, as described herein a universal framework library that might be suitable for CDR grafting is designed.

[0031] The present invention, in another embodiment, provides an isolated optimized human antibody comprising a heavy and light chain of a human antibody, whose overall sequence shares a low sequence similarity with that of a murine antibody, wherein a framework (FR) region in the human antibody possesses residues comprising a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or, in another embodiment, a positive B to M score and a positive human diversity score. In another embodiment, a positive CDR contact ratio score for particular residues in a FR is determined as the ratio between the number of CDR residues within 6A contact of an FR residue and the number of total residues within 6A contact of said FR residue and the positive CDR contact ratio score is a score of greater than or equal to 25%. In another embodiment, a positive B to M score for particular residues in a FR is determined as the frequency of FR residue mutations which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular FR residue in an antibody, which restored said binding. In another embodiment, a positive human diversity score is determined as a frequency of diversity at an FR residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0 45 Each possibility or combination thereof represents a separate embodiment, of the present invention and is exemplified herein (see Examples 3-6 below)

[0032] In one embodiment, the antibody further comprises a complementarity determining region (CDR) isolated from a muπne antibody In another embodiment, the CDR is grafted onto the human antibody Framework Region (FR) region In another embodiment, the human antibody's heavy chain's and light chain's overall sequences respectively share less than 61% identity with the murine antibody whose CDR sequences are used for grafting onto the human antibody framework region. In another embodiment, the similarity shared by the murine antibody with the human antibody template is 69% for the heavy chain and 76% for the light chain

[0033] The invention further provides, in one embodiment, a library of optimized human antibody templates for murine CDR grafting Each antibody template in the library comprises a heavy and light chain of a human antibody whose overall sequence shares less than 61% identity (76% similarity) with that of a murine antibody In another embodiment, the muπne 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 CDR of the murine monoclonal antibody onto the library of human antibody templates

[0034] In one embodiment, the term "library" refers to a set of universal framework region (FR) variants or antibody templates in any form, including but not limited to a list of nucleic acid or ammo acid sequences, a list of nucleic acid or ammo acid substitutions at variable positions, a physical library compπsing nucleic acids that encode the library sequences, or a physical library comprising the variant FR or antibody templates, 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. Such methods that may find use in the present invention are described or referenced in U.S. Pat No. 6,403,312, U.S. Ser No 09/782,004; U.S. Ser. No 09/927,790; U S Ser No 10/218,102; PCT WO 01/40091; and PCT WO 02/25588, all incorporated entirely by reference 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 the present invention for generating nucleic acids that encode the FR or antibody template libraries.

[0035] The present invention, in another embodiment, provides a process for preparing a library of affinity- optimized antibody templates to be used in another embodiment for the generation of affinity-optimized antibodies. In another embodiment, the invention provides for optimized antibody library generation, and antibodies and antibody fragments derived thereby, comprising universal framework region (FR) templates onto which an appropriate complementarity determining region (CDR) is grafted, preserving a desired affinity for a particular target, in a context, which in another embodiment, allows for minimal to no immune-mediated rejection of the molecule.

[0036] 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, percentage sequence "similarity" 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.

[0037] In another embodiment, "affinity optimization" refers to the optimization of the binding affinity of an antibody variable region including the selection of relevant acceptor framework amino acid positions to be altered. Alteration of donor CDR amino acid positions can be also considered an embodiment of the present invention, as will be required to produce affinity optimized antibodies and 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 structure or 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.

[0038] In one embodiment, the term "donor" refers to a parent antibody molecule or 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 structural or 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 donor CDRs confer binding affinity of the parent molecule onto the receiving molecule. It is sufficient that the donor is a separate and distinct molecule

[0039] As used herein, 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 IxICT⁶ M or IxIO^"7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslmkmg, two-hybrid assays and the like Appropriate controls can be used to distinguish between "specific" and "non-specific" binding.

[0040] 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 mtact light chain and a portion of the heavy chain, two Fab' fragments are obtained per antibody molecule;

(3) (Fab')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. [0041] In another embodiment, the antibodies or fragments as provided herein may 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, scFv-Fc fusions, 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 [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593- 596 (1992)].

[0042] 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 ammo 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 of 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), scFv-Fc fusions 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. Such terms are described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Molec. Biology and Biotechnoloqy: A Comprehensive Desk Reference (Myers, R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth Enzymol., 178:497-515 (1989) and in Day, E D., Advanced Immunochemistry, Second Ed , Wiley-Liss, Inc , New York, N Y (1990)

[0043] In one embodiment, the terms "isolated peptide" or "polypeptide" refers to an antibody as further described herein When in reference to any polypeptide of this invention, the term is meant to include native polypeptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the polypeptides more stable while in a body or more capable of penetrating into cells Such modifications include, but are not limited to N terminal, C terminal or peptide bond modification, including, but not limited to, backbone modifications, and residue modification, each of which represents an additional embodiment of the invention Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C A. Ramsden Gd., Chapter 17 2, F. Choplin Pergamon Press (1992) In one embodiment, a polypeptide is a full length protein or a variant of a known protein

[0044] In one embodiment the polypeptide of this invention may be an isoform of the isolated polypeptide In one embodiment, "isoform" refers to a version of a molecule, for example, a protein, with only slight differences to another isoform of the same protein In one embodiment, isoforms are produced from different but related genes, or in another embodiment, arise from the same gene by alternative splicing. In another embodiment, isoforms are caused by single nucleotide polymorphisms

[0045] In one embodiment the isolated polypeptide of this invention is a fragment of the native protein In one embodiment, "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, 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- termmal fragment. In another embodiment, the fragment is a C-termmal fragment. In one embodiment, the fragment of this invention is an mtrasequential 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-termmal functional fragment. In one embodiment, the fragment is a C-terminal functional fragment [0046] In one embodiment, an isolated polypeptide of this invention 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" 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

[0047] In another embodiment, the term "nucleic acid" refers to 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 mternucleoside (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.

[0048] 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. Each of these represents a separate embodiment of this present.

[0049] 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

[0050] 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-codmg for the protein of interest. The invention further provides DNA sequences which encode proteins similar to those encoded by sequences provided 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 provided 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.

[005I] 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 ammo 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)

[0052] In one embodiment, the polypeptide of this invention comprises an amino acid substitution In one embodiment, the ammo acid substitution is conservative. A "conservative amino acid substitution" is one in which the ammo acid residue is replaced with an amino acid residue having a similar side chain Families of ammo acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, argimne, 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, isoleucme, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g , threonine, valine, isoleucme) and aromatic side chains (e.g , tyrosine, phenylalanine, tryptophan, histidme) In another embodiment, the amino acid substitution is not a conservative one which results in enhanced activity of the mutated polypeptide compared to the native polypeptide

[0053] The polypeptides of this invention can be produced by any synthetic or recombinant process such as is well known in the art. 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

[0054] In one embodiment, the term "antibody template" refers to a vanable region, antibody fragment, or entire antibody minus the CDR or hypervanable region.

[0055] A "variant" of a polypeptide or protein, in one embodiment, refers to an ammo 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-bmdmg 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 ammo 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

[0056] 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 FRs satisfying the scoring criteria as provided herein, and therefore are viewed as acceptors In one embodiment, a diverse library is assembled by selecting acceptor framework positions that differ at the corresponding position compared to the donor framework and making a library population containing all of possible ammo acid residue changes at each of those positions together with all possible amino acid residue changes at each position within the CDRs of the variable region Grafting is accomplished, in another embodiment, by splicing a population of encoding nucleic acids for the donor CDR containing species representing all possible amino acid residues at each CDR position into a population of encoding nucleic acids for an antibody acceptor variable region framework which contains species representing all possible amino acid residue changes at the selected framework positions In certain embodiments, the resultant population encodes the authentic donor and acceptor framework ammo acid sequences as well as all possible combinations and permutations of these sequences with each of the 20 naturally occurring amino acids at the changed positions

[0057] In another embodiment, the affinity optimized antibody and antibody fragment libraries of this invention comprise a framework region, which in another embodiment is referred to as a "universal framework region" 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 (5 sup th ed 1991) Each variable domain typically has four FRs identified as FRl, FR2, FR3 and FR4 Through out this invention, we utilized the "Enhanced Chothia" numbering scheme in combination with the "Contact" definitions of CDRs in order to locate the FR regions This numbering scheme and CDR definition are proposed by Dr Andrew C R Martin's group at the Research Department of Structural and Molecular Biology, University College London (httpV/www bioinfo org uk/mdex html, specifically http //www biomf org uk/abs/#abhinum, last accessed on Nov 2^nd 2008) The main difference between the Chothia and the Enhanced Chothia numbering scheme is that the latter considers the structurally correct locations for indels in the FR regions Additionally, the "Contact" definition of the CDR relies on the analysis of the available crystal structures of complexed antibodies (MacCallum R M , Martin A C R and Thornton J T , J MoI Biol 262, 732-745) In another embodiment, upon combing the "Contact CDR" definition with the Enhanced Chothia numbeπng scheme, the light chain FR residues are positioned at residues 1-30 (LCFRl), 37- 45 (LCFR2), 56-88 (LCFR3), 102-110 (LCFR4) and the heavy chain FR residues are positioned 1-29 (HCFRl), 35A-46 (HCFR2), 59-92 (HCFR3), 102-110 (HCFR4) and these will represent the edges of the FR regions One skilled in the art will understand that the positions may vary based upon the numbeπng scheme and the CDR definition used In another embodiment, "FR" also refers to an antibody variable region compπsing 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

[0058] In another embodiment, the term "universal framework region" also refers to an antibody or antibody fragment, library of antibodies or antibody fragments, which comprise an FR prepared by the methods of this invention and onto which a donor CDR can be grafted as further described hereinbelow In another embodiment, the term "universal framework region" refers to a region possessing a combination of a positive CDR contact ratio score and a human diversity score or a combination of a positive B to M score and a positive human diversity score as described hereinbelow, and some of which are exemplified herein

[0059] In another embodiment, the antibody or antibody fragments of this invention or libraries comprising the same comprise a CDR grafted onto the Universal framework region, or FR templates of this invention The non- human antibody providing the CDR s is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor" Humamzation relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (Winter U S Pat No 5,225,539) In one embodiment, the majority of sequence variability occurs in the complementarity determining regions (CDRs) There are 6 CDRs total, three each per heavy and light chain, designated VH CDRl, VH CDR2, VH CDR3, VL CDRl, VL CDR2, and VL CDR3 The variable region outside of the CDRs is referred to as the framework (FR) region Although not as diverse as the CDRs, sequence variability does occur in the FR region between different antibodies Overall, this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens A number of high resolution structures are available for a variety of variable region fragments from different organisms, some unbound and some in complex with antigen The sequence and structural features of antibody variable regions are well characterized (Morea et al , 1997, Biophys Chem 68:9-16; Morea et al , 2000, Methods 20 267-279), and the conserved features of antibodies have enabled the development of a wealth of antibody engineering techniques (Maynard et al , 2000, Annu Rev Biomed Eng 2:339-376) For example, it is possible to graft the CDRs from one antibody, for example a murine antibody, onto the framework region of another antibody, for example a human antibody This process referred to in the art as humanization, enables generation of less immunogenic antibody therapeutics from nonhuman antibodies Fragments comprising the vanable region can exist in the absence of other regions of the antibody, including for example the antigen binding fragment (Fab) comprising VH-CHl and VL-CL, the variable fragment (Fv) comprising VH and VL, the single chain variable fragment (scFv) comprising VH and VL linked together in the same chain, as well as a variety of other vanable region fragments (Little et al , 2000, Immunol Today 21:364- 370)

[0060] In another embodiment, the term "structure" refers to a polypeptide, an antibody, or fragments thereof in complexed (bound) or uncomplexed (unbound) form as described herein

[006I] In one embodiment, the term "CDR" or "complementarity determining region" refers to ammo 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 262.732-7 '45 (1996) The amino acids of the CDRs of the variable domains were initially defined by Kabat, based on sequence variability, to consist of ammo 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 vanable domain (VL), using Kabat's numbenng system for ammo 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 vanable 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 vanable domain (VH), and ammo 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 ammo acid residues 26-35B (Hl), 50-65 (H2), and 95-102 (H3) in human VH and ammo acid residues 24-34 (Ll), 50-56 (L2), and 89-97 (L3) in human VL, based on Kabat's numbering system In another embodiment, the antibody sequence numbering follows a scheme called "Enhanced Chothia" This scheme was proposed Dr Andrew C R Martin's group at the Research Department of Structural and Molecular Biology, University College London (http.//www bioinfo org uk/index html, specifically http //www biomf org uk/abs/, last accessed on Nov 2ⁿ 2008) The mam difference between the Chothia and the Enhanced Chothia numbering scheme is that the latter considers the structurally correct locations for indels in the FR regions In another embodiment, the "Contact" definition is used to locate CDRs on Fv sequences This CDR definition relies on analyzing available crystal structures of complexed antibodies (MacCallum R M , Martin A C R and Thornton J T , J MoI Biol 262, 732-745) to determine the areas of the hyper-vaπable region responsible for contacting antigens Through out this invention, we combined the "Enhanced Chothia" numbeπng scheme with the "Contact" definition of CDRs Consequently, antibody CDRs through out this invention are comprised of amino acids residues 30-35B (Hl), 47-58 (H2), and 93-101(H3) in human VH and ammo acid residues 30-36 (Ll), 46-55 (L2), and 89-96 (L3) in human VL

[0062] Depending on the ammo 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 subumt structures and three-dimensional configurations of different classes of immunoglobulins are well known

[0063] In the design of the antibody and antibody fragments of this invention, in one embodiment, the antibody vanable region can be divided into three layers, comprising the CDR region, which in one embodiment is the functional part of the antibody responsible for antigen recognition, the near CDR layer which in another embodiment provides the structural support for the functional conformation of the CDR, and the far-CDR layer which in another embodiment maintains the antibody structure and has less contact with the CDR region In another embodiment, the Universal FRs and Universal FR templates of this invention comprise the near CDR layer and far-CDR layer, and a CDR having a desired specificity is grafted thereunto [0064] Methods of making antibodies and antibody fragments are known in the art (See for example, Harlow and Lane, Antibodies- A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference)

[0065] In another embodiment, the Universal FRs and Universal FR templates of this invention comprise a FR combinatorial structural platform that can support the functional conformations of the CDRs of any desired parental antibody while maintaining the specificity and affinity of the parental antibody In another embodiment, the CDR is derived from or isolated from a murine monoclonal antibody, and the Universal FRs and Universal FR templates of this invention are deπved at least in part from human sequences or primarily human sequences, such that the processes of preparation of the antibodies, antibody fragments and /or libraries of this invention represent antibody humamzation processes In another embodiment, the present invention utilizes structural and sequence information to build a consensus of near-CDR positions, and in another embodiment, far-CDR positions, responsible for supporting the functional conformation of CDRs.

[0066] In another embodiment, the affinity- optimized antibodies, antibody fragments and/or antibody templates of this invention are prepared by compiling structural and sequence information to arrive at a combination of a positive CDR contact ratio score and a human diversity score or a B to M score and a human diversity score, using the thresholds for each score as described herein, and some of which are exemplified herein that influence the design of a universal FR library In another embodiment, the affinity-optimized antibodies, antibody fragments and/or antibody templates of this invention are prepared by compiling structural and sequence information to arrive at a combination of a positive CDR contact ratio score and a human diversity score or a combination of a positive B to M score and a positive human diversity score that influence the design of a universal FR library

[0067] 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 ammo 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

[0068] Human or humanized antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J MoI. Biol., 227.381 (1991), Marks et al., J MoI Biol , 222.581 (1991)]. Further, and in one embodiment, phagemid vectors and kits for prepanng phage libraries known in the art are also used to arrive at the phage display libraries of humanized antibodies or variants thereof of the present invention. 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)] Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e g. mice m which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire This approach is described, for example, in U.S. Pat Nos 5,545,807; 5,545,806; 5,569,825, 5,625,126; 5,633,425; 5,661,016, and m the following scientific publications Marks et al , Bio/Technology 10, 779-783 (1992); Lonberg et al , Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al , Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev Immunol. 13 65-93 (1995)

[0069] In one embodiment, computer aided molecular design "CAMD" is a method that threads the mouse sequence into an antibody structure and points FR positions that are essential for CDR support The sequence of these positions are mutated in the human sequence back to the mouse sequence. Selection of the FR is based on the most homologous to parental (murine) human antibody sequence by which then the CDRs of the parental antibody are grafted into the acceptor human antibody However, CDR grafting alone does not retain the recognition of the parental antigen. Limitations of this methods include that the FR positions of the "Back to Mouse" mutations vary between different antibodies and is thus a case by case design; the method is a time- consuming one because multi-rounds of design refinements are required through experimental validation; specific binding to parental antigen is not completely restored because the inaccurate structural modeling that is usually done on homologous antibody structures. In another embodiment CAMD is a method for carrying out homology modeling

[0070] In one embodiment, in the method of the present invention the CDRs of the parental or donor antibody have low-sequence similarity or are non-homologous to the human antibody framework. FR positions that satisfy either a positive contact ratio score or a positive back to mouse mutation score and are their diversity score among human sequences is at least 0.45 are randomized into a library on the selected human scaffold. In another embodiment, the amino acid selections at these positions are only restricted to the most abundant substitutions in all the available human antibody sequence.

[007I] In one embodiment, compiling structural and sequence information involves assembling such information regarding multiple human antibodies of known specificity, and availing such information in a central location.

[0072] In one embodiment, the term "compiling" refers to a systematic storage of structural and sequence information regarding antibody regions such as variable heavy and light chains that comprise complementarity determining regions and framework regions as provided in the invention. The systematic storage of structural and 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 structural and 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, structural and 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 structural and 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.

[0073] In another embodiment, the term "compiling" refers to assembling structural or 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 structural and sequence software programs or databases, for example DNASTAR software, ClustalW, T-coffee, FASTA3, STRAP, ALIGN4D, NCBI, BLAST2SEQ, Sanger and PSI-BLAST or any additional database. The additional database can specifically contain arranged antibody information relating to structural and sequence information of CDRs and FRs relating to the scored information as described herein, and is to be considered as contemplated for use Further, by "compiling" it is meant that the structural or 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/antibodies/antibody fragments of this invention

[0074] In one embodiment, the term "structural information" refers to a representation of a conformation of a macromolecule in whole or in part at a resolution sufficient to determine the relative locations of two or more atoms The term can include, for example, a representation that can be used to determine the relative position of two or more atoms withm less than 50 A, less than 40 A, less than 30 A, less than 20 A, less than 10 A, less than όA, less than 3 A, less than 2 5 A or less than 2 A hi this regard, structural information refers to a collection of near-CDR framework residues that are responsible for CDR structural support required during antigen recognition These antibody framework residues are of interest, thus a "contact ratio", i e - the ratio between the CDR contact count of that framework residue and the total number of residue contacts it has within the antibody structure, was devised to identify these particular residues from the compiled data described herein In another embodiment of the present invention the compiled structural information yields a number of specific heavy and light chain Framework residues that make contacts with the CDR In another embodiment, these residues are ranked based on whether the "Contact Ratio" scores are predictive for experimental data available for antibody humanization as descπbed herein and as exemplified in Example 6

[0075] In another embodiment, structural information is derived using methods available in the art, for example X-ray crystallography The structural information gleaned from these methods is stored and scored based on the criteria provided herein to arrive at the Universal FR library

[0076] In one embodiment, compiling structural information is for the purpose of assigning a score for individual residues comprising an FR sequence, such that a Universal FR sequence or library of sequences may be derived Each position in such sequence or library of sequences reflects a choice, in part based on a score, dependent upon the structural information compiled [0077] In another embodiment, the affinity- optimized antibodies, antibody fragments and/or antibody templates of this invention are prepared by compiling structural and sequence information. Examples 1-2, below provide, in an embodiment of this invention, a manner of gathering structural and sequence information

[0078] In one embodiment, compiling structural and sequence information is for the purpose of assigning a score for individual residues comprising an FR sequence, such that a Universal FR sequence or library of sequences may be derived. Each position in such sequence or library of sequences reflects a choice, in part based on a score, dependent upon the sequence information compiled and/or structural information compiled

[0079] 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 withm 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 Universal FRs 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.

[0080] In another embodiment, scoring is reflective of values obtained, based on sequence information, where such scoring may be a function of identity and/or homology determinations, based on sequence alignments. 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 are a consequence of functional, structural, or evolutionary relationships between the sequences. In another embodiment, aligned sequences of nucleotide or ammo 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

[0081] 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 ammo 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.

[0082] 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 structure alignment are well known in the art, and include alignments based on sequence and structural homology, as described herein.

[0083] In one embodiment, the term "homology," "homolog" or "homologous" refers to sequence identity, or to structural identity, or 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 86% correspondence with the indicated sequence In another embodiment, the ammo 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 92% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% correspondence with the indicated sequence In another embodiment, the ammo 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 ammo 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 the amino acid sequence or nucleic acid sequence exhibits no more than 85% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 75% correspondence with the indicated sequence In another embodiment, the ammo acid sequence or nucleic acid sequence exhibits no more than 65-74% correspondence with the indicated sequence In another embodiment, the ammo acid sequence or nucleic acid sequence exhibits no more than 55 64% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 45-54% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 35-44% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 35-44% correspondence with the indicated sequence In another embodiment, the ammo acid sequence or nucleic acid sequence exhibits no more than 15-34% correspondence with the indicated sequence In another embodiment, the ammo acid sequence or nucleic acid sequence exhibits no more than 5-14% correspondence with the indicated sequence In another embodiment, the amino acid sequence or nucleic acid sequence exhibits no more than 0 1 4% correspondence with the indicated sequence In another embodiment, the term "non-homologous can be used interchangeably with the term "low sequence similarity"

[0084] In one embodiment, all possible pair-wise alignments for the sequences of the bound and unbound antibody structural datasets of the present invention are performed using BLAST2SEQ

[0085] In another embodiment, information is compiled from structural and sequence information regarding antibodies or fragments thereof in a bound or unbound conformation In another embodiment, information is compiled from structural and sequence information regarding antibodies or fragments thereof from any available source of such information [0086] In one embodiment, and as exemplified herein, abstracts from the Protein Data Bank that contain the word "antibody" are searched for structural information of unbound (uncomplexed) and bound (antibody/antigen complex) structures The selected structures are numbered according to the "Contact Definition" numbering scheme disclosed online (Website www biomfo org uk/abs last accessed on Nov 2^nd, 2008 or in another embodiment, other numbering are used, for example, Chothia or the Kabat numbering schemes as described (Sequences of Proteins of Immunological Interest 4"¹ Ed US Department of Health and Human Services Public Health Service (1987)) and Chothia and Lesk ((1987) J MoI Biol 186 651-663))

[0087] In another embodiment, compiling sequence and structural information regarding FRs is of such information for molecules across species, i e , such information is derived from human antibodies, murine antibodies, and any other species for which such information may be obtained

[0088] In one embodiment of the present invention and as exemplified herein, human and mouse antibody primary sequence alignment revealed an FR consensus composed of ammo acids similar in chemical property with regard to each other, however such FR positions in the respective human and mouse sequences exhibited low sequence diversity (Website http //www bioc unizh ch/antibody/Sequences/Germlines/SeqStat/mdex html, last accessed on Nov 2ⁿ , 2008) indicating that antibody CDRs may be structurally supported by a limited sequence diversity in the near CDR layer of the antibody FR, which in turn is reflective of information used in the compiling/scoring and assembly of the Universal FR antibody or antibody fragment library or template library as herein described

[0089] In one embodiment, sequence information is gathered by using an alignment program to perform all possible pair-wise alignments for the sequences of the bound and unbound antibody structural datasets In another embodiment, sequence redundancy in compiled datasets is removed, for example as described herembelow in Example 1

[0090] In another embodiment compiling such sequence and structural information reflects assembly of datasets such that sequence diversity is favored, for example, and In another embodiment, no two structures whose framework regions' heavy or light chains which are compiled withm such dataset share a sequence identity of more than 85% [0091] 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 In another embodiment, pairwise alignments are conducted using dot-matrix methods, dynamic programming, and word methods, as known in the art.

[0092] In one embodiment, multiple sequence alignment are conducted as an extension of pairwise alignments to incorporate more than two sequences at a time Multiple alignment methods try to align all of the sequences in a given query set and may identify conserved sequence regions across a group of sequences Such conserved sequence motifs can be used in conjunction with structural and mechanistic information to locate the catalytic active sites of enzymes Alignments are also used to aid in establishing evolutionary relationships by constructing phylogenetic trees It is understood that any of these methods can be used as will be appreciated by a skilled artisan.

[0093] 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, for example

[0094] In another embodiment, "dot-matrix" methods refer to an alignment approach which implicitly produces a family of alignments for individual sequence regions. 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. [0095] For purposes of classifying amino acids substitutions as conservative or nonconservative, ammo 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 ammo acids in the same class Non- conservative substitutions constitute exchanging a member of one of these classes for a member of another

[0096] In another embodiment, "dynamic programming" methods are 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 ammo 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.

[0097] 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.

[0098] In another embodiment, "word methods" are used, which comprise their implementation in database search tools such as the FASTA and the BLAST family Mount DM (2004 Bwinformatics: 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, non-overlapping 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

[0099] In one embodiment, any structural alignment method existing in the art can be used as understood by a skilled artisan For example, the DALI method, the SSAP method or the combinatorial extension method

[00100] In one embodiment, the DALI method, or distance matrix alignment, is a fragment-based method for constructing structural alignments based on contact similarity patterns between successive hexapeptides in the query sequences Holm L, Sander C (1996) "Mapping the protein universe" Science 273 595-603 PMID 8662544 This method can, In another embodiment, generate pairwise or multiple alignments and identify a query sequence's structural neighbors in the Protein Data Bank (PDB)

[00101] In one embodiment, the SSAP (sequential structure alignment program) is a dynamic programmmg- based method of structural alignment that uses atom-to-atom vectors in structure space as comparison points It has been extended since its original description to include multiple as well as pairwise alignments, (Taylor WR, Flores TP, Orengo CA (1994) "Multiple protein structure alignment" Protein Sa 3: 1858-70 PMID 7849601 ) and has been used in the construction of the CATH (Class, Architecture, Topology, Homology) hierarchical database classification of protein folds (Orengo CA, Michie AD, Jones S, Jones DT, Swindells MB, Thornton JM (1997) "CATH-a hierarchic classification of protein domain structures" Structure 5: 1093-108) The CATH database can be accessed at CATH Protein Structure Classification

[00102] In one embodiment, the combinatorial extension method of structural alignment generates a pairwise structural alignment by using local geometry to align short fragments of the two proteins being analyzed and then assembles these fragments into a larger alignment (Shindyalov IN, Bourne PE (1998) "Protein structure alignment by incremental combinatorial extension (CE) of the optimal path' Protein Eng 11: 739-47 PMID 9796821) Based on measures such as rigid body root mean square distance, residue distances, local secondary structure, and surrounding environmental features such as residue neighbor hydrophobicity, local alignments called "aligned fragment pairs" are generated and used to build a similarity matrix representing all possible structural alignments within predefined cutoff criteria A path from one protein structure state to the other is then traced through the matrix by extending the growing alignment one fragment at a time. The optimal such path defines the combinatorial-extension alignment A web-based server implementing the method and providing a database of pairwise alignments of structures in the Protein Data Bank is located at the Combinatorial Extension website.

[00103] In one embodiment, a structure-based sequence alignment is used to search for a highly diverse hit library. In another embodiment, this method is used for comparing various multiple sequence alignments m the absence of any detectable sequence homology (Sauder I M, Arthur J W, Dunbrack R L Ir (2000) Proteins 40, 6-22). In another embodiment, the multiple structure alignment directly yields the corresponding multiple sequence alignment. In yet another embodiment, these closely related structures are used as structural templates for sequence threading to generate the multiple sequence alignment profile (Jones D T (1999) J MoI Biol 1999, 797-815). In one embodiment, methods combining multiple sequence and structure alignments have been reported to annotate the structural and functional properties of known protein sequences (Al Lazikani B, Sheinerman F B, Honig B (2001) PNAS 98, 14796-14801).

[00104] In another embodiment the human antibody templates of the present invention comprise non-CDR regions derived from a single human antibody that share low sequence similarity with the parental antibody. In another embodiment, such antibody template backbone will be derived from a human antibody, which shares little or low sequence identity with a murine antibody, whose CDR is desired for grafting onto the antibody template being constructed. In another embodiment, the identity shared by the murine antibody with a human antibody template is 51% for the heavy chain and 61% for the light chain or less. In another embodiment, the similarity shared by the murine antibody with a human antibody template is 69% for the heavy chain and 76% for the light chain or less

[00105] In certain embodiments, the non-CDR regions derived from a single human antibody, share less than 61% identity (76% similarity) with the murine parental antibody.

[00106] In one embodiment a percent sequence identity threshold is used to filter out undesired structures In one embodiment the threshold is 85% where no two structure's framework region's heavy or light chains share a sequence identity more than 85%. In another embodiment, no two structure's framework region's heavy or light chains share a sequence identity more than 75%. In another embodiment, no two structure's framework region's heavy or light chains share a sequence identity more than 65%. In another embodiment, no two structure's framework region's heavy or light chains share a sequence identity more than 55%. In another embodiment no two structure' s framework region' s heavy or light chains share a sequence similarity of more than 85% In another embodiment no no two structure's framework region's heavy or light chains share a sequence similarity of more than 75%

[00107] In selecting a framework (e g. human germline), in another embodiment, a number of criteria are employed (although employing selection criteria is not necessary to understand or practice the present invention). For example, one may select frameworks that are 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 human frameworks that are frequently used in the human population). One could also select framework derived from population analysis from a variety of ethnic populations, e.g arrive at FRs commonly found in European Caucasian, different FR backbones in Africans, different FR backbones derived from analysis of Afncan American, amongst other racial backgrounds. By selecting these frameworks, when armed with the invention as described herein, a skilled artisan can apply the scoring to the FR residues.

[00108] In one embodiment, the antibody/antibody fragments/antibody templates of this invention and libraries of this invention comprise FRs, which have been selected, or comprise a universal FR, which is based on the compiling of structural and sequence information to arrive at such FRs, where scoring of such sequences or specific residues within such sequences is utilized. In one embodiment, the scoring includes a "back to mouse" or "B to M" score for particular residues in a Framework Region (FR) of multiple antibodies from which sequence and structural information is compiled, where "B to M" refers to back-mutation of selected acceptor framework residues to the corresponding donor residues in order to regain affinity that is lost in the initial grafted construct. In certain embodiments, the B to M score is arrived at by gathering and using sequence information from sequence databases, index searches, journal publications, or a combination thereof to arrive at "B to M" mutations that determine the score In another embodiment, collecting the "B to M" data from available humanization literature shows that there are essential FR positions that need to be back-mutated to the murine sequence in order to restore the parental antibody binding ability to its target antigen. Further, these back mutations yield a series of humanized antibodies, which in the literature have been rendered more active/desirable, based on select mutation of these particular residues to approximate that of a murine antibody homologous to the human Such B to M score will necessarily be positive for inclusion of such information within a dataset/library/antibody/process of this invention. The dataset can be ever expanded and updated to reflect newly accumulated data for positions within a given FR template library [00109] In one embodiment, the term "backmutation" or "back to mouse mutation" refers to a process in which some or all of the somatically mutated amino acids of a human antibody are replaced with the corresponding germline residues from a homologous germline antibody sequence. The heavy and light chain sequences of a human antibody of the invention are aligned separately with the germline sequences m the VBASE database to identify the sequences with the highest homology. VBASE is a comprehensive directory of all human germline variable region sequences compiled from published sequences, including current releases of GenBank and EMBL data libraries. The database has been developed at the MRC Centre for Protein Engineering (Cambridge, UK) as a depository of the sequenced human antibody genes (website: http://www.mrc-cpe.cam.ac.uk/vbase- intro.php?menu=901). Differences in the human antibody of the invention are returned to the germline sequence by mutating defined nucleotide positions encoding such different amino acids The role of each amino acid thus identified as a candidate for backmutation should be investigated for a direct or indirect role in antigen binding, and any amino acid found after mutation to affect any desirable characteristic of the human antibody should not be included in the final human antibody. To minimize the number of amino acids subject to backmutation, those amino acid positions found to be different from the closest germline sequence, but identical to the corresponding amino acid in a second germline sequence, can remain, provided that the second germline sequence is identical and co-linear to the sequence of the human antibody of the invention for at least 10, preferably 12, amino acids on both sides of the amino acid in question. Backmutation may occur at any stage of antibody optimization.

[00110] An accumulation of literature regarding antibody humanization can be gathered online (website: http://people.cryst.bbk.ac.uk/~ubcg07s/selhum.html, last accessed on Nov 2^nd, 2008). In certain embodiments, this website provides a source of publications that contains mutational data regarding crucial mouse (parental) sequences on the humanized antibodies that restore binding, enhance binding to the corresponding antigen, or any other functional or physical characteristic of the antibody that is desired, e.g. stability, lysis, opsonization, activity, lack of being a cryoprecipitant, immunogenicity. These B to M mutations are gathered, In another embodiment, from online or any other sources that yield the necessary information in usable format, as will be readily appreciated by a skilled artisan. In another embodiment, specific residues, based on Enhanced Chothia numbering, as described herein, are mutated to residues more typically found in murine antibodies, and results in restoration of antibody binding affinity to its original target antigen or, in one embodiment, in antibodies with greater affinities for the target antigen. Such selection or scoring is reflective of an occurrence of at least 4 such substitutions being found in any of the various databases or combinations thereof as an indication of importance to humanization success. Those positions having more than 4 counts of reoccurring B to M mutations are regarded as expeπmental hotspots essential for participating in antigen recognition of the parental mouse antibody In another embodiment, Back to Mouse mutation hotspots are essential for supporting mouse CDR orientation while recognizing the antigen In another embodiment, the B to M score is arrived at by determining the frequency of Framework Region (FR) residue mutation which results in restoring binding of a humanized antibody to a target In another embodiment the B to M mutation is a conservative amino acid substitution In certain embodiments of the present invention, it is understood that a skilled artisan can use any method available in the art or as described herein to arrive at a positive B to M score In another embodiment, a positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restores binding to an antigen In another embodiment it is a score of greater than 10 mutations at a particular Framework Region (FR) residue in an antibody, which restores said binding In another embodiment it is a score of greater than 15 mutations at a particular Framework Region (FR) residue in an antibody, which restores said binding In another embodiment, it is understood by a skilled artisan that the B to M score can be updated, revised or modified so as to accommodate for a development of more efficient libraries

[00111] In one embodiment, the antibody/antibody fragments/antibody templates of this invention and libraries of this invention comprise FRs, which have been selected, or comprise a universal FR, which is based on the compiling of structural and sequence information to arrive at such FRs, where scoring of such sequences or specific residues within such sequences is utilized In one embodiment, the scoring includes a complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies from which sequence and structural information is compiled Such score will necessarily be positive for inclusion of such information within a dataset/hbrary/antibody/process of this invention

[00112] In another embodiment, the CDR contact ratio score is arrived at by compiling structural and sequence information to arrive at specific amino acid residues within a given antibody molecule or fragment thereof within an FR region, which positions are crucial for CDR contact, CDR support, CDR conformation and/or proper antigen recognition Such positioning choices are reflected in arrival at a "contact ratio" score, which refers to the ratio between the complementaπty determining region (CDR) contact count of a framework residue and the total number of residue contacts it has within the antibody structure In certain embodiments, this normalization reflects correction for the side chain size difference among the natural twenty ammo acids

[00113] In one embodiment, the CDR contact ratio score for a particular residue is a value assigned to specific sites in a framework (FR) of an antibody or antibody template molecule, where the FR residue contacts a CDR residue, or is in close proximity thereto, or contact other residues which impact the CDR conformation, or In another embodiment, the overall antibody conformation or In another embodiment, the fragment conformation, which allows for desired characteristics of the antibody, for example, greater antibody affinity or antibody stability, or any desirable characteristic as herein descπbed In another embodiment, the "contact ratio" is arrived at by compiling structural information and sequence information as will be appreciated by a skilled artisan and as descπbed herein to determine the ratio between the CDR contact count of a FR residue and the total number of residue contacts it has withm an antibody structure In certain embodiments, the ratio is between the number of CDR residues withm 6A contact of a Framework Region (FR) residue and the number of total residues within 6A contact of that FR residue In another embodiment it is withm 50- 40 A, 30-20 A, 20-10 A , within 5 -withm 2 A In another embodiment, the contact ratio analysis, described herein, remains constant regardless of the similarity threshold used In certain embodiments, the contact ratio can be arrived at by using any structural and sequence information available in the art, as will be understood by a skilled artisan, or as specified herein above It is to be understood by a skilled artisan that the contact ratio score can be updated, revised or modified so as to accommodate for a development of more efficient libraries

[00114] In one embodiment, a side by side comparison or analysis of CDR contact ratio scores of the FR residues m in a large non-redundant dataset of bound and unbound antibody structures determines the FR positions that are ultimately responsible for CDR structural support, which is required during antigen binding and/or recognition In another embodiment, the contact ratio scores are averaged on the available non-redundant antibody structural data, and thus proves to be advantageous over CMAD which entails the analysis to be carried out on a case by case basis

[00115] In one embodiment, the positive CDR contact ratio score is a score of greater than or equal to 25% In another embodiment, the positive contact ratio score is a score of greater than or equal to 35% In another embodiment, it is a score of greater than or equal to 45% In another embodiment, it is a score of greater than or equal to 55% In another embodiment, it is a score of greater than or equal to 65% In another embodiment, it is a score of greater than or equal to 75% In another embodiment, it is a score of greater than or equal to 85% In another embodiment, it is a score of greater than or equal to 95% In another embodiment, it is a score of greater than or equal to 96% In another embodiment, it is a score of greater than or equal to 97% hi another embodiment, it is a score of greater than or equal to 98% In another embodiment, it is a score of greater than or equal to 99% In another embodiment, it is a score of 100% [00116] In one embodiment, a framework consensus composed of amino acids that are chemically similar are identified, in one embodiment, by aligning human and mouse antibody primary sequences

[00117] In one embodiment, the amino acid diversity at each column of the multiple alignments (Figure 1) is computed as the Shannon Entropy of the amino acid distribution by using the following formula.

where P/ is the probability of amino acid / in each column of the multiple alignment It is to be understood by a skilled artisan that other methods of determining diversity that are available in the art can also be used, for example see (Caffrey et al , Protein Sci 2004 Jan;13(l):190-202)

[00118] In one embodiment the unlimited conformational geometries of the antibody's CDRs are structurally supported, by a limited sequence diversity in the near-CDR layer of the framework. In one embodiment, the antibody/antibody fragments/antibody templates of this invention and libraries of this invention comprise FRs, which have been selected, or comprise a universal FR, which is based on the compiling of structural and sequence information to arrive at such FRs, where scoring of such sequences or specific residues within such sequences is utilized In one embodiment, the scoring includes a reflection of observed human diversities at near-CDR positions

[00119] In another embodiment, the human diversity score is determined as a frequency of diversity at a FR residue, which, in one embodiment, is present in human antibody sequences. In another embodiment, FR positions that satisfy either of the CDR contact ratio score or the B to M score are considered for later stages of the design if their diversity score among human sequences is at least 0.45. In another embodiment, there are "outliers" which refers to amino acid choices that have a positive B to M score at these positions but are very conserved among human sequences, based on sequence information gathered from sequence databases, index searches, journal publications, or a combination thereof, are imported from mouse antibody sequences that already contain the human choice m order to restore antigenic binding capacity. In another embodiment, these mutations are few in numbers as compared to the human diversity sequences introduced in the library design and in another embodiment these outliers are not determined using the KABAT numbering scheme but rather the "Enhanced Chothia" scheme as described and exemplified herein. In yet another embodiment, FR structural and expenmental hotspots in the library design are included if their human sequence diversity is greater than a 0 1 threshold. In another embodiment, said positive human diversity score is a score of greater than 0 4. In another embodiment, said positive human diversity score is a score of greater than 0.8 In another embodiment, said positive human diversity score is a score of greater than 1 0 In another embodiment, said positive human diversity score is a score of greater than 0.45. In another embodiment, mouse sequence diversities are included at positions that have high back to mouse counts but human antibody sequences are conserved

[00120] In one embodiment, this invention makes use of a combination of at least 2 positive scores as provided herein, selected from the B to M score AND human diversity score, OR contact ratio score AND diversity score. In another embodiment, reliance solely upon Back to Mouse mutations suffers the limitations that the FR positions of the "Back to Mouse" mutations vary between different antibodies (necessitating a time-consuming case by case design). Similarly, multiple rounds of experimental validation are required, which again is a time consuming process Moreover, such methods may also result in binding comparable to the parental niAb being restored, which may not reflect the arrival at the optimized antibody characteristic, as herein described Applicants have exemplified herein, in Example 5, the lack of concordance between antibodies obtained, solely based on B to M mutation (as a means of generating antibodies, which are humanized, yet bind a specific target) versus those obtained by combining scores as herein provided, for example, based on contact ratio scores.

[00121] In one embodiment, once the structural and sequence information is compiled and the scores of the present invention are arrived at, the process for preparing a library of affinity-optimized antibody templates further comprises assigning any of the two combinations 1) a positive CDR contact ratio score AND a positive human diversity score OR 2) positive B to M score AND a positive human diversity score. Thresholds for each score are described hereinabove and some of which are exemplified herein, see example 1 and 5. In another embodiment, assigning a combination of two of the scores described herein will enable a skilled artisan to arrive at a universal FR library It is to be understood by a skilled artisan that the score thresholds can be modified, updated or revised for producing a more efficient affinity- optimized antibody templates onto which parental or donor CDRs can be grafted, and will reflect changes to such scores based on additional structural and/or sequence information available for antibodies being incorporated into the libraries, for construction of additional Universal FR templates/libraπes/antibodies/antibody fragments, as will be appreciated by the skilled artisan.

[00122] In another embodiment, scoring is reflective of statistical analysis conducted, and in one embodiment, any statistical method that is appropriate may be utilized as will be appreciated by a skilled artisan In another embodiment, such analysis is conducted to assess, determine the significance of, modify, revise and/or arrive at a contact ratio score, B to M score or human diversity score. For example, Chi-square tests, T-tests, mean, standard error and other readily available statistics tests can be applied to arrive at these scores. In another embodiment, correlations are computed using the spearman correlation coefficient accompanied with a p-value In another embodiment, contact consensus analysis (Mean and standard error) for all framework positions is computed from all the structural data. In another embodiment, Profile Hidden Markov models or HMM statistics are used. In another embodiment, Profile Hidden Markov Models or HMM are statistical models of the primary sequence consensus of a given sequence or sequence alignment family. In another embodiment, the sequence family is defined as the multiple sequence alignment resulting from the corresponding multiple sequence and/or structure alignment In another embodiment, the formal probabilistic basis underlying HMM makes it possible to use Bayesian probability theory to guide the setting of the scoring parameters based on the profile of aligned sequences. In another embodiment, this same feature also allows the HMM to use a consistent approach, using the position-dependent scores, to score the alignment for both amino acids and gaps In another embodiment, these features in HMM make it a powerful method to search for remote homologues compared to the traditional heuristic methods (Eddy S. R (1996) Curr Opin Struct. Biol 6, 361-365). In another embodiment, the pattern m the primary sequence is detected by the pattern recognition algorithms and therefore is used to pull out more members related to the target sequence (when one sequence is used) or sequence profile (when multiple sequence alignment is used), hi another embodiment, to capture the higher order correlation in a sequence, or the interactions between amino acids in three-dimensional space, the multiple sequence alignment resulting from multiple structural alignments is used.

[00123] In one embodiment, a FR variant nucleic acid library of the present invention is generated by designing a series of overlapping nucleic acid oligonucleotides including the selected human templates with selected positions diversified according to the diversity score desired, as provided herein, to graft CDRs onto the selected human antibody scaffold. In another embodiment the oligonucleotides are annealed to each other via Overlapping Polymerase Chain reaction (PCR), a method well known in the art, to assemble the genes required for the humanization design. In another embodiment, the overlapping oligonucleotides contain degenerate codon choices to arrive at residues which satisfy the diversity score desired that cover the desired amino acid diversities in the FR positions selected for randomization and will allow a combination of two positive scores to be achieved, as described herein.

[00124] In another embodiment, the Fab library of the present invention is generated by designing a series of overlapping nucleic acid oligos are designed and synthesized including the selected human templates with selected positions diversified according the design, and CDRs of C225 (typically 50-90 base pairs in length, Table 13). To each variable domain, 4 of the oligos (group A) to the 5'-termmus and 4 oligos (group B) to the 3 '-terminus are separately mixed and annealed, followed by Deep Vent® mediated extension to fill the gaps To join the nicks Taq DNA hgase which works at 45⁰C is used so that no polymers form in low temperature Expected size of bands (A band) in group A and group B (B band) are recovered, in one embodiment, by agarose gel isolation and DNA extraction, which are routine in the art Band A and B were then mixed equal- molecularly to splice the whole variable length, which was then amplified by standard PCR Humanized VL and VH DNA are sequentially cloned into a phagemid vector that uses gene El for display including those known and readily available in the art, including but not limited to, pComb3 and its derivatives pCANTAB pHEN etc Briefly, humanized VL pool is cloned via ApaLI - Xhol, yielding a size of 2E6 cfu (colony- forming unit) sub-library Then VH pool was cloned into the sub-library via Sfil - BstEII, resulted final Fab library with estimated size of 5E9 cfu Standard protocol is used for the phage library rescue and panning, as described in examples below

[00125] In another embodiment, the methods of preparing the FR variant nucleic acid library include but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the FR Site-directed mutagenesis is in certain embodiments, a method for preparing substitution variants This technique is well known in the art (see, e g Carter et al , (1985) Nucleic Acids Res 13 4431-4443 and Kunkel et al , (1987) Proc Natl Acad Sci USA 82: 488 492, both of which are hereby incorporated by reference) Each antibody in the library comprises in its own discrete embodiment, a Framework Region (FR) region selected by combining and thresholding two of three scores described herein, these being In another embodiment, a positive CDR contact ratio score, a positive B to M score, and a positive human diversity score The method further comprises, in one embodiment, assembling a library, whereby, in another embodiment, each antibody in the library of human antibody templates comprises a Framework Region (FR) region possessing residues having a combination of two of a positive complementarity determining region (CDR) contact ratio score, a positive B to M score or a positive human diversity score as provided herein

[00126] In one embodiment, the process for preparing a library of affinity-optimized antibody templates comprises compiling structural and sequence information regarding multiple human antibodies of known specificity The process further comprises assembling a library of human antibody templates In another embodiment, the library can be a nucleotide, protein or phage library, or a vector library comprising the nucleotides that code for the desired templates [00127] In another embodiment, the library is a nucleic acid library, 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. According to this aspect of the invention, and in another embodiment, the invention provides a library of affinity-optimized antibodies of known specificity prepared according to a process of the invention. In another embodiment, the libraries/methods of the present invention arrive at a universal FR library, which in turn is used to generate a library of antibodies with known specificity by swapping in the desired CDR from a parental antibody.

[00128] In one embodiment, a library of sequences of framework regions of human Ig heavy chain variable domains is prepared for diversification, by using the Kabat numbering system (see E. A. et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, National Institutes of Health, USA (5.sup.th ed. 1991), where each murine Immunoglobulin (Ig) heavy chain and/or light chain variable domain framework amino acid sequence and murine Ig CDR amino acid sequence is aligned (e.g., by aligning electronically or manually) with all or a portion (subset) of the human heavy and/or light chain variable domain framework amino acid sequences and CDR amino acid sequences included in the Kabat database using an alignment program existing in the art or as described herein below. In some cases, it is desirable to elect a predetermined number of mismatches to ensure that the final recombinant heavy chain framework region possesses a desired degree of similarity to the donor heavy chain framework amino acid sequence. For example, if the number of mismatches is chosen to be greater than or equal 0.35 times the number of residues in a given framework, then the resulting recombinant heavy chain framework amino acid sequence is less than 85% identical to the donor heavy chain framework amino acid sequence.

[00129] 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.

[00130] 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 another embodiment 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), and are commercially available As such, it is to be understood by a skilled artisan that any phage display system known in the art can be used to arrive at the affinity-optimized antibody (or variant thereof) libraries of the present invention. These phage display systems are thus encompassed for use m the methods of and to arrive at the compositions of the present invention.

[00131] An assembly cell is a cell m 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.

[00132] In one embodiment, phage display is used to create the FR variant library. In another embodiment, the method of preparing a phage display FR variant library comprises, in another embodiment, the steps of modifying a phagemid vector for cloning, assembling VH and VL variable FR regions, followed by sequencing analysis, sequential cloning of VL and VH into the phagemid vector, and building a large size library

[00133] It is to be understood by a skilled artisan that a phage library comprising the antibodies or variants thereof provided herein is routinely created by any method known in the art for generating such a library. In one embodiment the 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 coll 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.

[00134] In one embodiment, the phages from the FR variant library are rescued and panned against EGFR as described in example 9 and representing an embodiment of the invention. In another embodiment, best binders are selected and their sequence is analyzed. In another embodiment, the best binders are cloned into a phagemid vector to build a large library comprising the FR variants

[00135] Introduction of nucleic acid encoding humanized antibody, antibody templates, FR libraries, or parental CDR into target cells can also 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, polyamme acids, hydrogel, polyvinyl pyrrohdone, ethylene- vmylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevmylacetate 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

[00136] 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 canonic 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

[00137] In one embodiment, nucleotide sequences are 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, ampicillm resistance, kanamycm 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 another embodiment of the present invention expression constructs contain both positive and negative selection markers

[00138] 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

[00139] 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.

[00140] 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.

[00141] 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, inhibiting or preventing

[00142] In another embodiment, the present invention comprises compositions comprising polynucleotides, vectors, polypeptides, antibodies and/or fragments thereof as herein described, capable of inhibiting to and/or host cells. In another embodiment, the present invention comprises methods of use of a polynucleotide, vector, antibodies and/or fragment thereof as herein described and/or compositions comprising the same in treating, inhibiting or preventing.

[00143] The invention also provides transformed cells and progeny thereof into which a nucleic acid molecule encoding humanized antibody, antibody templates, FR libraries, or parental CDR has been introduced by means of recombinant DNA techniques m 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.

[00144] In one embodiment, the term "transformed" refers to 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 earned 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.

[00145] 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 m vitro (e.g., in solution or in solid phase), in cells or in vivo.

[00146] 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.

[00147] 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 drag, 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 puromycm, 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

[00148] 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-thymidme 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 fhioxanthme

[00149] 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-dividmg 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)

[00150] In one embodiment, phagemid vectors for use m the invention include any available m the art suitable for the production of the antibodies/antigen-bmding fragments of the present invention and include but are not limited to phagemid vectors pEGFP-Nl, RC-CMVb, pRC-CMV, pCGMT-lb, pCGMT, 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; 6,472,147, 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.

[00151] 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. hi another embodiment of the present invention the construct also include, a virus, a plasmid, a bacmid, a phagemid, a cosmid, or a bacteriophage.

[00152] An affinity optimized 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, e.g., an antibody phage display library, as exemplified herein in examples 7 and 8. These and similar methods available in the existing art of generating FR or antibody template libraries can be used along with a target antigen, to thereby isolate antibody library members that bind specifically to the target antigen. Kits for generating and screening phage display libraries are commercially available (see for 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).

[00153] 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, hi another embodiment, 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.

[00154] In one embodiment, the term "target antigen" refers to the molecule that is bound specifically by the variable region of a given antibody. A target antigen may be a protein, carbohydrate, lipid, or other chemical compound. In one embodiment, the term refers to, but is not limited to antigens such as TP53, PTEN, PDLIM 1,SPRX, NUCBl, and PSCA which are prostate cancer pathway- specific tumor suppressor genes; FOLHl, KDR, PSIPl, EGFR, E7RBB2, CCKBR, XLKDl, MMP9, TMPRSS2, AGR2, PRSS8, MUCl, LGALS8, CD164, CXCR4, CXCR7, ABCB5, NRPl, STEAPl, HPN, MET, PTGER3, CLDN3, CLDN4, NCAM2, EDNRB, FLTl, PECAMl, BDKRB2, CD151, QSCN6, ERG, PCNA, EPCAM, and MADlLl which are cell surface proteins expressed by some cancer cells; HSPAlA, HSPBl, SERPINHl, HSPA5, TRAl, MICB, PSMAB4, UBE2C, STIPl, HSPDl, and UBQLNl are proteins involved in innate immunity; EIF4G1 ALOX15, PTGSl, RPL23, RPS14, ELACl, EIF3S3, TOP2A, RPS6KA1, ACPP, KLK3, FASN, RPL30, and ENOl which are proteins involved in cell metabolism; CCNBl, CCNDl, CCNA, CDKN2A, CUL4A, BIRC5, MYC, ETS2, BCL2, BCLG, TP53BP2, GDF15, RASSFl, AKTl, MDM2, PIMl, SH3GLB1, HIMP2, HEVIP3, KHDRBSl, PCNA, and CAV3, are cell cycle or apoptosis-related proteins; and E6 and E7 which are human papillomavirus antigens. HIPl, BRD2, AZGPl, COVAl, MLHl, TPD52, PSAP, MIBl, H0XB13, RDHI l, HMGA2, ZWINT, RCVl, SFRP4, SPRRlB, HMGA2, HIP2, and HEYL which were also found to be cancer- associated, or any other cancer-related antigen existing in the art, an isolated and/or purified thereof, a fragment thereof, an immunogenic fragment thereof, a fusion protein thereof, a domain thereof, or an epitope thereof.

[00155] In another embodiment, recombinant antibody libraries are 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 m 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

[00156] In one embodiment, a library is screened using one or more cell-based or m 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 [00157] Antibodies 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 unpuπfied, 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 caloπmetry, 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 chromogemc, fluorescent, luminescent, or isotopic labels.

[00158] 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, a yeast or bactenophage, 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

[00159] 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

[00160] 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)

[00161] 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 deπvatives such as selective phage infection (Malmborg et al , 1997, J MoI Biol TTi 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 bacteπa (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, JAm Chem Soc 124, 538-543).

[00162] 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

[00163] 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).

[00164] 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). 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 is carried out using the methods of Kunkel (infra), for example to introduce a modified CDR or a random sequence to substitute for a CDR. In another embodiment, light and heavy chain variable regions are humanized separately, and then co-expressed as a humanized variable region. In another embodiment, humanized variable regions make-up the variable region of an intact antibody. [00165] In one embodiment, the process of generating an affinity-optimized antibody further comprises grafting the CDRs of the parental murine antibody of desired specificity onto each antibody template of the library as described herein and as will be appreciated by a skilled artisan. 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 said antibodies in said library formed Li another embodiment the method further comprises identifying an antibody having the highest binding affinity for said 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 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 hybndoma 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 hybndoma 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 hybndoma cell The hybndoma 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 hypoxanthme guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthme, ammopteπn, 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, munne 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 descnbed for the production of human monoclonal antibodies (e g., Kozbor, J. Immunol., 133: 3001 (1984), herein incorporated by reference) [00166] 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 histmol 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-omithme, DFMO (McConlogue (1987) In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed )

[00167] 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

[00168] DNA 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

[00169] In one embodiment, a final 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 chromatofocusmg 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 resm if a GST fusion is employed, Ni⁺ 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

[00170] In another embodiment, antibody fragments are prepared by proteolytic hydrolysis of the antibody or by expression in E coll 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 pepsm 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

[00171] Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR) CDR peptides ( 'minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells See, for example, Larrick and Fry, Methods, 2: 106 10, 1991

[00172] In another embodiment, antibodies or antibody fragments are isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al , Nature, 348 552554 (1990) Clackson et al , Nature, 352 624-628 (1991) and Marks et al , J MoI Biol , 222 581-597 (1991) that descπbe the isolation of murine and human antibodies, respectively, using phage libraries Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, BioTechnology, 10" 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (e g , Waterhouse et al , Nuc Acids Res , 21 2265-2266 (1993)) Thus, these techniques, and similar techniques, are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies Further and m another embodiment, the DNA is modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (e g , U S Pat No 4,816,567, and Morrison, et al , Proc Nat Acad Sci USA, 81 6851 (1984), both of which are hereby incorporated by reference), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulm polypeptide Typically such non-immunoglobulm polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody compπsing one antigen-combmmg site having specificity for an antigen and another antigen-combining site having specificity for a different antigen

[00173] 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 prepared are known in the art For example, many such sequences are disclosed in Kabat et al (1991) supra and in the "Vbase" human germhne sequence database, administered by the MRC Centre for Protein Engineering (Cambridge, UK) (website http //www mrc cpe cam ac uk/vbase intro php?menu=901)

[00174] 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

[00175] 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 calorimetπc dyes or molecules such as biotm 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 [00176] 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, a 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.

[00177] 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, caloπmetry, 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

[00178] 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 m-human 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 FcγRIIa 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. 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

[00179] In one embodiment, the chimeric antibody cetuximab binds Epidermal Growth Factor Receptor (EGFR) EGRF is differentially expressed in many human cancers and EGFR is a major anticancer therapeutic target. The primary function of cetuximab is to block the ligand -stimulated EGFR signaling

[00180] 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 , VoI : 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, VoI : 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 VoI : 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.

[00181] 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 VoI : 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

[00182] 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.

[00183] In one embodiment, the anti-EGFR antibody is selected from Cetuximab (Erbitux), Panitumomab (Vectibix), Zalutumomab (HuMax-EGFR), Matuzumab (EMD72000), and an anti-EGFRvIII antibody.

[00184] In one embodiment of the invention, the anti-EGFR antibody is Cetuximab. In another embodiment, the anti-EGFR antibody is any antibody selection from the following list of antibodies or an Fab, ScFv, or functional fragment thereof, that are represented by accessions number ABG27073, ABX79402, ABX79401, ABX79400, ABX79399, ABX79398. ABX79397, ABX79396, ABX79395, ABX79394, ABX79393, ABX79392.

[00185] 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.

[00186] In another embodiment, the EGFR antibodies are selected from chimerized, humanized, fully human, and single chain antibodies derived from the murine antibody 225 described in U.S. Pat. No. 4,943,533 to Mendelsohn et al, The EGFR antibody can also be selected from the antibodies described in U.S. Pat. No. 6,235,883 to Jakobovits et al., U.S. Pat. No. 5,558,864 to Bendi et al., and U.S. Pat. No. 5,891,996 to Mateo de Acosta del Rio et al.

[00187] The Cetuximab antibody or C225 is a genetically engineered chimeric murine-human monoclonal antibody directed against the EGF receptor antigen. Cetuximab is the antibody called described in Naramura et al. (1993) Cancer Immunol. Immunotherapy, Vol.: 37, pages 343-349. Cetuximab is marketed as Erbitux.

[00188] EGF (Epidermal Growth Factor) and EGF Receptor (EGFR) have been described and characterized for example in frit L A X et al. (1988) "Localization of a Major Receptor-Binding Domain for Epidermal Growth Factor by Affinity Labeling" Molecular and Cellular Biology, VoI 8 No. 4, pages 1831-1834 and references therein; and [0022]Graham CARPENTER (1979) "Epidermal Growth Factor" Ann. Rev. Biochem. Vol.: 48, pages 193-216. The term "EGFR" or "EGF-Receptor" according to the present invention encompasses full length and native EGFR and truncated versions thereof, in particular EGFRvIII. In another embodiment, the term "EGFR" or "EGF-Receptor" according to the present invention is to be understood as meaning human full length and native EGFR

[00189] Cetuximab is indicated (1) in combination with irinotecan in patients with EGFR-expressing colorectal cancer who have failed prior irinotecan therapy, (2) in combination with radiotherapy as first line treatment for locally advanced squamous cell carcinoma of the head and neck, and (3) in combination with cisplatin as first line treatment for recurrent of metastatic squamous cell carcinoma of the head and neck Further, the L2987 human lung carcinoma has been characterized as positive for the EGF receptor and borderline levels of antitumor activity for cetuximab have been described. The GEO human colon carcinoma is also known to be EGF receptor positive and amongst the responsive tumor models to cetuximab and gefitmib, a small molecule anti-EGFR inhibitor

[00190] One process envisioned by the invention is exemplified herein in Example 7-8, where CDRs from the mouse donor antibody (M225) are grafted onto an optimized human antibody template library It will be readily apparent to a skilled artisan that the methods described herein can be used to humanize any other antibodies.

[00191] In another embodiment of the present invention, the human antibody template framework is derived from one 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 yet another embodiment, the human antibody template framework is derived from one or a combination of the light chain variable region Kappa III (VK III) or from any other light chain variable region as will be appreciated by a skilled artisan In another embodiment, these human V_H subgroup III and V_L-kappa subgroup III consensus sequences are selected as frameworks, respectively, to humanize a parental antibody such as a murine antibody as described in Example 5 and 7. Thus, consensus sequences known in the art, as exemplified for human V_H subgroup III or V_L-kappa subgroup I, can be chosen as acceptor frameworks for producing humanized antibody in accordance with the invention. In one embodiment, the invention provides an isolated humanized antibody selected from a library as described herein

[00192] In these embodiments, one or more non-human CDRs are grafted onto the universal FR template library. Also allowed is human CDR grafting, which is not optimal in terms of stability, affinity, or other desired characteristic, but which the human CDR can be grafted onto the FR libraries of the invention. In one embodiment, a universal, framework region of this invention comprises a series of antibodies, or antibody fragments, or antibody or antibody fragment templates, which will comprise or onto which a CDR of a parent antibody will be grafted. In yet another embodiment, a universal human framework with mutations only in residues critical for structural support is used as the framework for all antibodies to be humanized because of its low sequence homology to the framework sequence(s) of the donor or parent antibodies. This universal, human framework can then accept one or more CDR sequences. The library may comprise multiple backbones, or multiple versions of a similar backbone, with point mutations to address the scoring issues of the present invention.

[00193] The antibodies 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, hi 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

[00194] In one embodiment, the invention also provides a kit for preparing a library of human antibody templates In this regard, the kit comprises a library of polynucleotides encoding human antibody templates comprising Framework Region (FR) regions possessing residues comprising a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score as described herein above.

[00195] In another embodiment, the invention provides a kit for preparing a library of human antibody templates, and comprises, in another embodiment, a polynucleotide that encodes a murine antibody fragment which comprises a complementarity determining region (CDR) region and whose grafting onto the library of human antibody templates is desired. In another embodiment, the invention provides reagents for grafting the nucleic acid-encodmg a murine antibody fragment and comprises complementarity determining region (CDR) regions on the library of human antibody templates that are in the library of polynucleotides that encode the human antibody templates that comprise Framework Region (FR) regions

[00196] In one embodiment, the kit comprises a vector comprising the polynucleotides encoding human antibody templates, parental CDRs, FR library, or humanized antibody of the invention, or in another embodiment, the kit comprises bacteriophages comprising the polynucleotides encoding human antibody templates parental CDRs, FR library, or humanized antibody of the invention. The kit may have the particular backbone and the enzymes/reagents for mutating particular FRs based on scoring information, which information may be provided, optimized for particular CDRs, e.g. kits for antibodies recognizing a target antigen with choice of 2, 3 or multiple backbones, as will be determined by a skilled artisan to assemble particular libraries Further the kits may have the template libraries that are optimized for particular parental CDRs.

[00197] In another embodiment, 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 humanized antibody, antibody template, FR library, or parental CDRs. hi another embodiment, a kit includes a nucleic acid encoding humanized antibody, antibody template, FR library, or parental CDRs. 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.

[00198] In additional embodiments, a kit includes a label or packaging insert including instructions for expressing a humanized antibody or a nucleic acid encoding a humanized antibody, antibody template, FR library, or parental CDRs 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

[00199] 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

[00200] 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.

[00201] In another embodiment, "symptoms" may be any manifestation of a disease or pathological condition as described hereinabove.

[00202] 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.

[00203] 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.

[00204] 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.

[00205] This invention provides, in another embodiment, optimized humanized antibodies as herein described, including in another embodiment, specifically antibodies prepared as products of the processes as described herein, wherein such process comprises a method which selects appropriate FR templates as described herein, possessing the appropriate score, wherein such score is reflective of a positive CDR contact ratio and a positive human diversity score or the a positive B to M score and a positive human diversity score.

[00206] The humanized antibodies of the invention, including subsequences, modified forms, multimers and nucleic acids encoding them, can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for administration to a subject in vivo or ex vivo, and for providing therapy for a physiological disorder or condition treatable with a humanized antibody.

[00207] In one embodiment the compositions of this invention comprise a polypeptide of this invention, 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

[00208] In one embodiment, the route of administration is parenteral. In another embodiment, the route may be intra-ocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intraarterial, vaginal, rectal, mtratumoral, 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

[00209] 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

[00210] 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

[00211] 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.

[00212] For liquid formulations, pharmaceutically acceptable earners 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 salme 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.

[00213] In one embodiment, a composition of or used in the methods of this invention are 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 diglycendes, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc In another embodiment, the pharmaceutical preparations are 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 are also combined where desired with other active agents, e g , vitamins

[00214] In one embodiment, 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

[00215] 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.

[00216] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a steπle 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.

[00217] 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 polyetheylene 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, and 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.

[00218] 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.

[00219] 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.

[00220] The present invention's humanized antibodies, 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

[00221] 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.

[00222] Additional pharmaceutical formulations appropriate for the compositions for administration in the methods of the invention are known in the art (see, e g , Remington's Pharmaceutical Sciences (1990) 18^th ed , Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12^th ed , Merck Publishing Group, Whitehouse, N J.; and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co , Inc , Lancaster, Pa , (1993)) The pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage "Dosage unit form" in one embodiment refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier or excipient

[00223] It is to be understood that any amino acid sequence, whether obtained naturally or synthetically by any means, exhibiting sequence, structural or functional homology to the polypeptides described herein, are considered part of this invention.

[00224] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning' A laboratory Manual" Sambrook et al , (1989); "Current Protocols in Molecular Biology" Volumes 1-111 Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al , "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U S Pat. Nos 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J E , ed (1994); "Current Protocols in Immunology" Volumes I-III Coligan J E , ed (1994), Stites et al (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shngi (eds), "Selected Methods in Cellular Immunology", W H Freeman and Co , New York (1980), available immunoassays are extensively described in the patent and scientific literature, see, for example, U S Pat Nos 3,791 932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3 867,517, 3,879,262 3,901,654, 3,935,074, 3,984,533; 3,996,345, 4,034,074; 4,098,876, 4,879,219; 5,011,771 and 5,281,521, "Oligonucleotide Synthesis" Gait M J , ed (1984), "Nucleic Acid Hybridization" Hames, B D , and Higgins S J , eds (1985), "Transcription and Translation" Hames, B D , and Higgins S I , eds (1984), "Animal Cell Culture" Freshney, R I , ed (1986), "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B , (1984) and "Methods in Enzymology" VoI 1-317, Academic Press; "PCR Protocols A Guide To Methods And Applications", Academic Press San Diego, CA (1990), Marshak et al , "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), Biotechnol Bioeng 1999 Oct 5,65(1) 1-9 Prediction of antisense oligonucleotide binding affinity to a structured RNA target Walton SP, Stephanopoulos GN, Yarmush ML, Roth CM , Prediction of antisense oligonucleotide efficacy by in vitro methods O Matveeva, B Felden, A Tsodikov, J Johnston, B P Moma, J F Atkms, R F Gesteland & S M Freier Nature Biotechnology 16, 1374 - 1375 (1998), all of which are incorporated by reference as if fully set forth herein Other general references are provided throughout this document The procedures therein are believed to be well known in the art and are provided for the convenience of the reader All the information contained therein is incorporated herein by reference

[00225] In one embodiment, polypeptides of the present invention are administered as part of a vaccine hi another embodiment, the term vaccine is to be understood to encompass any immunomodulating composition, and such vaccines comprise an adjuvant, an antigen, an immuno-modulatory compound or a combination thereof, in addition to the polypeptides of this invention

[00226] In another embodiment, the adjuvant includes, 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 another embodiment, (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 mterleukms (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 cytosme, (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 coll heat labile enterotoxm ("LT"), or detoxified mutants thereof such as the K63 or R72 mutants, (U) cholera toxm ("CT"), or diphtheπa toxin ("DT") or detoxified mutants of either ; (V) double-stranded RNA; (W) monophosphoryl lipid A mimics, such as ammoalkyl glucosaminide phosphate derivatives e g RC-529], (X) polyphosphazene (PCPP); or (Y) a bioadhesive such as esteπfied hyaluronic acid microspheres or a mucoadhesive such as crosshnked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose

[00227] 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

[00228] 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 In another embodiment, 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 [00229] 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.

[00230] 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.

[00231] In another embodiment, the compositions of this invention will consist essentially of a polypeptide/polynucleotide/vector as herein described In another embodiment, the term "consisting essentially of refers to a composition whose only active ingredient of a particular class of agents, is the indicated active ingredient, however, other compounds may be included which are involved directly in the therapeutic effect of the indicated active ingredient. In another embodiment, the term "consisting essentially of refers to a composition whose only active ingredient of targeting a particular mechanism, or acting via a particular pathway, is the indicated active ingredient, however, other compounds may be included which are involved directly in the therapeutic effect of the indicated active ingredient, which for example have a mechanism of action related to but not directly to that of the indicated agent. In another embodiment, the term "consisting essentially of refers to a composition whose only active ingredient is the indicated active ingredient, however, other compounds may be included which are for stabilizing, preserving, etc the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. In another embodiment, the term "consisting essentially of may refer to components which facilitate the release of the active ingredient. In another embodiment, the term "consisting" refers to a composition, which contains the active ingredient and a pharmaceutically acceptable carrier or excipient

[00232] 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. [00233] In one embodiment, the compounds of the invention are administered acutely for acute treatment of temporary conditions, or are administered chronically, especially in the case of progressive, recurrent, or degenerative disease. In one embodiment, one or more compounds of the invention are administered simultaneously, or in another embodiment, they are administered in a staggered fashion. In one embodiment, the staggered fashion is dictated by the stage or phase of the disease

[00234] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringers and fixed oils Intravenous vehicles include fluid and nutrient replemshers, 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 liquid carriers, particularly useful 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

[00235] In another embodiment, the compositions of this invention 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, tπethyl 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.

[00236] Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g , corn starch, pregeletanized starch), a sugar (e.g , lactose, manmtol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g , polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof

[00237] 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, hi 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.

[00238] 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

[00239] 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, solubihzers or other substances.

[00240] 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 ammo 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, trimethylamme, 2-ethylamino ethanol, histidine, procaine, and the like.

[00241] 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, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid

[00242] In one embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the therapeutic compound is released over a period of time after administration. Controlled- or sustamed-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 hi 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 peπods 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 hi another embodiment, compositions of the invention are administered prior to development of gross symptoms

[00243] In yet another embodiment, the pharmaceutical composition of this invention can be delivered in a controlled release system For example, the antibody or portion thereof 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 can be used In yet another embodiment, a controlled release system can be 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 any controlled release system known in the art

[00244] In another embodiment, the antibodies and antibody portions of the present invention are administered by a variety of methods known in the art, where in another embodiment, for many therapeutic applications, the 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 hi 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

[00245] 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

[00246] Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody or antibody portion of the invention is co-formulated with and/or co-administered with one or more additional therapeutic agents.

[00247] 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).

[00248] In another embodiment, the term "full-length antibody" refers to the structure that constitutes the natural biological form of an antibody. In most mammals, including humans, and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains V_L and C_L, and each heavy chain comprising immunoglobulin domains V_H, Cγl, Cγ2, and Cγ3. In each pair, the light and heavy chain variable regions (V_L and V_H) are together responsible for binding to an antigen, and the constant regions (C_L, Cγl, Cγ2, and Cγ3, particularly Cγ2, and Cγ3) are responsible for antibody effector functions. In some mammals, for example in camels and llamas, full-length antibodies may consist of only two heavy chains, each heavy chain comprising immunoglobulin domains V_H, Cγ2, and Cγ3. By "immunoglobulin (Ig)" herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include but are not limited to antibodies Immunoglobulins may have a number of structural forms, including but not limited to full-length antibodies, antibody fragments, and individual immunoglobulin domains including but not limited to V_H, Cγl, Cγ2, Cγ3, VL, and C_L

[00249] 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 vaπant of the wild type sequence.

[00250] 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

[00251] 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 an antigen 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

[00252] 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.

[00253] 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.

[00254] In one embodiment, optimized antibodies of the present invention are 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 mtraorgan (e.g. pancreas, prostate or mammary gland) injection of human tumor cells or fragments of donor patients

[00255] In embodiments, antibodies of the present invention are 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

[00256] In one embodiment, the testing of antibodies includes 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.

[00257] In another embodiment, 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 is 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.

[00258] In another embodiment, the antibody is administered with one or more immunomodulatory agents. 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 to¹ non-steroidal antiinflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, lndomethacm, ketoralac, oxaprozin, nabumentone, sulmdac, tolmentm, 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, CDIl, 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, IFNα, IFNβ, IFNγ, 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-I, LFA-3, MHC, selectins, TGFβ, TNFα, TNFβ, 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, glutar aldehyde, gold, hydroxychloroquine, leflunomide, malonomtriloamides (e g leflunomide), methotrexate, minocycline, mizoπbme, mycophenolate mofetil, rapamycin, and sulfasasazine

[00259] In an alternate embodiment, antibodies of the present invention are administered with a cytokine. By "cytokine" in one embodiment, 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 bovme growth hormone; parathyroid hormone; thyroxine; insulin; promsulm; relaxm; 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-alpha and -beta; mullenan-inhibitmg substance; mouse gonadotropin-associated peptide; mhibin; activin; vascular endothelial growth factor; integπn; thrombopoietm (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as mterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukms (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-alpha or TNF-beta; 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

[00260] 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 (C max), the time to reach Cmax (Tmax), the area under the plasma concentration-time curve from time 0 to infinity [AUC(O-mf)] and apparent elimination half-life (T₁/₂) Additional measured parameters could include compartmental analysis of concentration-time data obtained following 1 v administration and bioavailability

[00261] 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.

[0249] 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.

[0250] 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 Hodgkm 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 bihary 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), skm (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 hemagiopeπcytoma)

[00262] In one embodiment, the term "about" refers to a value within 10% of the underlying parameter (i e , plus or minus 10%), and is sometimes a value within 5% of the underlying parameter (i e , plus or minus 5%), a value sometimes withm 2 5% of the underlying parameter (i e , plus or minus 2 5%), or a value sometimes within 1% of the underlying parameter (i e , plus or minus 1%), and sometimes refers to the parameter with no variation Thus, a distance of 'about 20 nucleotides in length" includes a distance of 19 or 21 nucleotides in length (i e , within a 5% variation) or a distance of 20 nucleotides in length (i e , no variation) In another embodiment

[00263] It is to be understood that reference to any publication, patent application or issued patent is to be considered as fully incorporated herein by reference in its entirety [00264] It is to be understood that this invention provides compositions, kits and uses of any combination of any agents as described herein, and such combinations represent embodiments of this invention

[00265] It is to be understood that any method of this invention, as herein described, encompasses the administration of a compound as herein described, or a composition comprising the same, to the subject, in order to treat the indicated disease, disorder or condition. The methods as herein described each and/or all may further comprise administration of an additional therapeutic agent as herein described, and as will be appreciated by one skilled in the art.

[00266] It is to be understood that any assay for measuring a particular activity which is modulated by the therapeutic compound may be employed, as a means of determining the efficacy of the compound, in one embodiment, optimal loading of the compound, in another embodiment, timing and dosage, in another embodiment, or a combination thereof

[00267] The following examples are intended to illustrate but not limit the present invention.

EXAMPLES

EXAMPLE 1

Compiling Antibody Structural Datasets

[00268] Structures were extracted from the Worldwide protein Data Bank (PDB) whose abstracts contained the word "Antibody' After manually eliminating antibody-ligand and unusual antibody (e g camelids, scFv) structures, 125 unbound antibodies and 119 antibody/antigen (bound) complexes were accumulated whose structures were determined via X-ray crystallography.

[00269] Software "Abnum" (Antibody Numbering) from the website http://www.bioinfo.org.ulc/abs/abnum/ (Dr. Andrew CR. Martin at the University College of London, Last visited November 2^nd, 08) was utilized to assist with the numbering of the sequences of the selected structures according to their "Enhanced Chothia" numbering scheme (Chothia with structurally corrected FR indels) (Reference of this application is MoI Immunol 2008 Aug; 45 (14):3832-9. Epub 2008 M 9). Pair- wise alignments for the sequences of the bound and unbound antibody structural datasets were conducted using the National Center for Biotechnology Information (NCBI) BLAST2SEQ program. To eliminate sequence redundancy from both datasets, the following graphical scheme was implemented: If structure A and structure B had a framework sequence identity exceeding 85% and the X-ray resolution of A was higher than B; then, an arrow was drawn to connect A to B according to the following manner A-> B If A-> B -^ C, then, A and C are kept while B was eliminated If A->B -> C and A~> C, then both B and C were eliminated

[00270] After determining the similarity networks in both datasets, redundant structures were eliminated based on the rules discussed above

[00271] This resulted in the placement of 62 structures in the bound antibody (Table 1) dataset and 75 structures in the unbound dataset (Table 2) Hence, there are no two structures within these datasets whose framework regions' heavy or light chains share a sequence identity of more than 85%

EXAMPLE 2

Compiling "Back to Mouse" Antibody Datasets

[00272] The website http://peoplexryst bbk ac uk/~ubcgO7s/selhum html compiles accumulated details of 48 publications regarding antibody humamzation, including reports from as early as 1997 Mutational data for maintaining crucial mouse (parental) residues may be derived therein, regarding antibodies subjected to various humamzation protocols, where mutations in certain residues from the humanized form back the residue associated with the mouse antibody restored binding to the corresponding antigen Such mutations are referred to herein as "Back to Mouse" mutations, and are assumed essential for supporting mouse CDR oπentation, preserving antigen recognition The database compiled roughly 221 "Back to Mouse" or "B to M" mutations Additionally, 137 humamzation publications that are not included in the depositary mentioned above were extracted from pubmed Of those 137 records, 41 publications related to humamzation by CDR grafting were manually filtered 440 back to mouse mutational data points were extracted from this new set of publications Our final back to mouse mutational data set is comprised of 221 light chain and 440 heavy chain FR back mouse mutational data points

EXAMPLE 3

Determining CDR Contacts [00273] Two residues are considered in contact if they contain two atoms within όΛ from each other. Because residues of the antibody framework that supports the CDR were of interest, the term "Contact Ratio" was defined to be the ratio between the CDR contact count of that framework residue and the total number of residue contacts it has within the antibody structure. This normalization is essential to correct for the side-chain size difference among the natural twenty amino acids For the bound antibody structural dataset the structure of the antigen was not included in the contact count

EXAMPLE 4

Estimating Diversity

[00274] Human VH (3099) and VL (2528) sequences were extracted from the KabatMan database (www.bioinfo.org uk/abs/). After generating multiple alignments for each sequence category, the human amino acid distribution at each FR position was determined. The amino acid diversity at each column of the multiple alignments was computed as the Shannon Entropy of the ammo acid distribution at that position The following Equation was used to compute Shannon's Entropy: -Σ P₁ log P₁ ; Λ>0

I

Where P_t is the probability of ammo acid i in each column of the multiple alignment.

EXAMPLE 5

Library Criteria and Design

[00275] The anti-EGFR monoclonal chimeric antibody, cetuximab (Erbitux) blocks ligand-stimulated EGFR signaling. The Fab form of cetuximab, C225 was chosen as an antibody humanization and optimization was desired. Grafting of the C225 onto a human framework, optimized by the methods of the invention was desired Toward this end, a series of templates were prepared, onto which the CDR was grafted.

[00276] Toward this end, a framework derived from the most abundant human subclasses was desired. Since the VH subgroup III (VHIII) often confers comparatively higher expression, stability and monomer formation to overall Fab or scFv in Escherichia coll regardless of the VL subclass used, a VH III germline clone (Acc# M99960, which belongs to IgHV3-23) and VK III germline clone (Acc# Ml 5038, which belongs to IgVK3-20) were chosen for the sake of stable expression in the format of Fab in prokaryotic systems [00277] In addition to stable expression, the choice in heavy and light chains reflected low sequence similarity with that of the parental strain from which the CDR was derived, i.e the mouse Fab C225

[00278] In terms of library construction and expression, the FR variant incorporating a candidate mouse CDR was expressed, displayed and screened by phage display technology, as described Baca (Baca, M., et al. (1997) J Biol Chem 111, 10678-10684; 19. Krauss, J., Arndt, M. A., Martin, A. C, Liu, H., and Rybak, S. M. (2003) Protein Eng 16, 753-759; 21. Villani, M. E , Morea, V , Consalvi, V., Chiaraluce, R , Desiderio, A , Benvenuto, E., and Donim, M. (2008) MoI Immunol 45, 2474-2485).

[00279] A side-by-side comparison of the CDR Contact Ratio scores of the FR residues in bound and unbound antibodies was evaluated to determine differences in their impact on the FR positions that are responsible for CDR structural support required during antigen recognition.

[00280] Table 1: listing of 62 bound antibody structures whose FR amino acids share an identity of less than or equal to 85%. PDB Id stands for the PDB identification codes for each of the structures. In Chain Id column, the letters to the left of the colon represent the PDB chain codes of the Fab, and the letters to the right represent the PDB chain codes of the antigen.

[00281] Table 2, listing of 75 unbound antibody structures whose FR amino acids share an identity of less than or equal to 85% are presented. PDB Id stands for the PDB identification codes for each of the structures. Chain Id column includes the PDB chain identification codes of the Fab.

[00282] No significant differences between the CDR Contact Ratio scores of the FR residues in both antibody structural datasets was found, however, hence FR positions with CDR Contact Ratio scores greater than or equal to 25% were selected. Thus, FR positions were considered as CDR structural support hotspots if at least 25% of their contacts were with residues of the CDRs.

[00283] Additionally, a dataset of Back to Mouse mutations was collected from published antibody humanization literature, as described above. Based on the collective data obtained, all the Back to Mouse mutated positions were tabulated and their frequencies counted as an indication of importance to humanization success. Those positions having more than 14 counts of reoccurring Back to Mouse mutations were regarded as experimental hotspots that are required to retain antigen recognition of the parental mouse antibody. [00284] The framework of the variable regions was determined using the KabatMan Server, including the "Enhanced Chothia" numbering scheme and the "Contact CDR" definition. The size of the structural datasets used in the "Contact Ratio" analysis after filtering out all the structures with sequence identities above 75% and 85% respectively is displayed in the following Table (Table 3):

[00285] Table 3:

[00286] A CDR contact was then established as being a framework residue that is within 6A of any CDR residue. To normalize the results by residue size, all framework residues "Contact Ratio" were computed. Contact consensus analysis (Mean and standard error) for all framework positions was computed from all the structural data and results are presented in Figure 2, for bound and unbound Fv heavy (A) and light (B) FR3 . No real difference was observed in terms of contact score, when evaluating bound versus unbound antibodies.

[00287] Traditionally, FR positions essential for the sensitivity of the humanized antibody were retained exclusively to their murine sequence. Figure 3 compares the possible amino acid substitutions along murine and human antibody sequences. Figure 3 shows a reasonable similarity among the most frequent amino acid substitutions when comparing murine antibody sequences to those of humans. Due to this similarity one can choose to concentrate on the human diversity while building the humanization library in order to decrease the mouse content in the humanized antibody sequence.

[00288] To arrive at a universal template library, thresholding was used as a selection criterion for specific positions for diversifying the FR. Such thresholding included incorporation of FRs that satisfies either Filter 1 : Contact Ratio of greater than or equal to 25% and a human diversity score greater than or equal to 0.45 or Filter 2: "Back to Mouse" count of greater than or equal to 14 and human diversity score greater than or equal to 0.45 . The CDR Contact Ratio, Back to Mouse mutational frequency, and diversity analysis for each light FR fragment is demonstrated in Figures 4-7; that of the heavy FR fragments are in Figures 8-11

[00289] Table 4 depicts the FR positions that passed the filters for the humanization library design. The "Filter" column includes information regarding the selection method: Filter 1 means the position passed the CDR Contact Ratio Back > 25% and the human diversity > 0.45 filters; Filter 2 means the position passed the Back to Mouse > 14 and the human diversity > 0.45 filters. KabatMan sequences were utilized to generate the "Diversity" column. This column includes the most observed human amino acid substitutions at these positions. In some positions we also included the most observed mouse (Underlined) substitutions. If the diversity column does not include amino acid substitutions; then, the corresponding position was not randomized for one of the following reasons. K and T dominate the amino acid choices at positions L45 and L97 respectively in both the mouse and human light chain KabatMan sequences. Hence, these positions were not randomized. In order to maintain a manageable library size, it was decided not to diversify these positions because their Back to Mouse frequency was among the lowest for all the selected heavy chain FR positions. Hence, it was decided not randomize in order to control library size. Position H69 in the KabatMan sequences is dominated by the amino acid I and L in human and mouse sequences, respectively. However, the L choice (mouse) at H69 is not observed in the human sequences, hence, it was decided not to include those choices in randomization in order to decrease mouse content in our library. The resulting library has an estimated size of approximately 6x10⁶

[00290] Table 4 shows the resultant diversity and size of the library based on the design presented herein. Specific amino acids are highlighted, representing the human diversity at indicated sites, taken from frame work repair analysis, with the total library size estimated at being 6xlO⁶.

Enh.

Cho thia Filter Diversity Enh. Chothia Filter Diversity

Ll 1 E D H27 1, 2 F Y

L2 1 I L H28 1, 2 N S T

L28 1 S D G N H29 1, 2 F L

L29 1 L V H35A 1 §§

L45 1 ** H35B 1 §§

L56 1 S T H37 1 I V

L58 1 I V H60 1 SS

L71 1, 2 A F Y H66 2 K R

L97 1 ** H67 2 A F V

H69 1 H

H71 1, 2 A R V

H72A (H73) 2 KN T

H75 (H78) 2 A F L V

H102 1 L V Y

[00291] Reliance solely upon Back to Mouse mutations suffers the limitations that the FR positions of the "Back to Mouse" mutations vary between different antibodies (necessitating a time-consuming case by case design). Similarly, multiple rounds of experimental validation are required, which again is a time consuming process Moreover, such methods may also result in binding comparable to the parental mAb being restored.

[00292] In addition, FR structural and experimental hotspots were included in the library design if their human sequence diversity was higher than a 0 45 threshold. Those different constraints resulted in selecting seven and ten positions in the antibody light and heavy FR chains, respectively. Three heavy and one light chain FR positions (H28, H66, H67, L71), however, exhibited high Back to Mouse counts, yet antibody human sequences were fairly well conserved at these positions. Consequently, the mouse sequence diversities were included at these four positions. Additionally, the mouse options Asp, Asn and GIy were included to the diversity at position L28 (Filter 1 only; Table 4) because there was not enough human diversity at this position. The estimated size of the final library design is around 6xlO⁶. Thus, one embodiment of the proposed methods of this invention including the grafting of CDRs a non-human antibody onto a non-homologous human antibody template, where the FR of the template in particular comprises a consensus sequence optimized by the scoring methods described herein, which results in the selection of templates optimized for FR positions lending crucial CDR conformational support.

EXAMPLE 6

Integrating Structural, Sequential and Experimental Data to Arrive At A Universal Framework

[00293] It was of interest to determine whether the "Contact Ratio" scores were indeed predictive for experimental data available for antibody humanization. Toward this end, positions overlapping between the computational method and available experimental data were plotted (Figure 13), including positions whose "Back to Mouse" scores were not zero (Figure 13). An r ~ Spearman rank order correlation coefficient was derived, and proved of particular significance for the heavy chain (rH = 0 6 (P-value ~ 3.4 10-5)) This result showed that a CDR contact consensus exists m antibody framework residues, which does not vary when comparing bound and unbound states of antibodies. Heavy chain framework residues that contact CDRs tended to be essential for restoring the binding of humanized Abs Upon integrating structural, sequential and experimental data, a universal framework library was designed suitable for CDR grafting.

EXAMPLE 7

Deriving Particular Antibodies Using the Universal Framework

[00294] To demonstrate the applicability of the Universal Framework, the M225 antibody was "humanized" The M225 antibody is a muπne antibody directed against the epidermal growth factor receptor (EGFR) Selections for humanization were conducted as described in Example 5, with the resulting proposed humanization sequence as depicted in Figure 14 A heavy chain template was designed (Figure 15A), and a series of heavy chain fragments with putative hot spots was derived (Figure 15B). Similarly, a light chain template was designed, and a series of fragments with putative hot spots was derived, as well (Figure 15C) Comparing the derived humanization template with that of the C225 antibody for both heavy and light chain demonstrated less than 61% identity there between A final library was derived (Figure 16), where the heavy chain diversity was 15552, whereas the light chain diversity was 384, arriving at a total library size of 6xlO⁶ It is possible to arrive at greater diversity, as well by degenerate codon usage. Multiple methodologies might be pursued for construction of the libraries as described herein. For example, and representing one embodiment of the invention, phage display methodology may be undertaken. A phagemid vector may be modified for cloning The heavy and light chains are assembled by standard methodology and sequence analysis is conducted. The individual light and heavy chains are then cloned into the phagemid vector, and a large sized library is prepared. The phage is rescued and panned against the appropriate antigen, in this example, EGFR Binding affinity and sequence analysis on individual fragments/antibodies is assessed

EXAMPLE 8

Fab Library Design, Panning and Evaluation

[00295] A series of overlapping nucleic acid oligos were designed and synthesized including the selected human templates with selected positions diversified according the design, and CDRs of ^C225 (typically 50-90 base pairs in length, Table 13). To each variable domain, 4 of the oligos (group A) to the 5 '-terminus and 4 oligos (group B) to the 3'-terminus were separately mixed and annealed, followed by Deep Vent® mediated extension to fill the gaps. To join the nicks Taq DNA ligase which works at 45⁰C was used so that no polymers that would form in low temperature. Expected size of bands (A band) in group A and group B (B band) were recovered by agarose gel isolation and DNA extraction Band A and B were then mixed equal-molecularly to splice the whole variable length, which was then amplified by standard PCR Humanized VL and VH DNA were sequentially cloned into modified phagemid vector, for example, pCANTAB, pComb3, etc Briefly, humanized VL pool was cloned via ApaLI - Xhol, yielding a size of 2E6 cfu (colony-forming unit) sub-library Then VH pool was cloned into the sub-library via Sfil - BstEII, resulted final Fab library with estimated size of 5E9 cfu Standard protocol was used for the phage library rescue and panning, as described in example. Table 1 lists 62 bound antibody structures whose FR ammo acids shared an identity of less than or equal to 85% In the "Chain Id" column, the letters to the left of the colon indicate the chain identities of the antibody, and to the right indicate the chain identities of the antigen.

[00296] Table 5 . Designed oligos for the humanization of C225 using universal framework library.

#: number of diversity combinations

[00297] Panning of humanized M225 Fab phage display library was followed by standard solid phase panning protocol with minor modification. Briefly, in the first round, 10 μg/ml of EGFR/Fc was coated to immunotube. Fab phage aliquot with a titer of about 1.OxIO¹² cfu was used for initial panning. The amount of antigen was reduced to 1 μg/ml in the second round. In the third round, to remove lower affinity binders, lμM of free EGFR/Fc was added to the amplified 2^nd round phage solution while it was incubated with 1 μg/ml immobilized EGFR/Fc. The competition incubation lasted three hours at room temperature before washing. [00298] Well-isolated single colonies from 2^nd and 3^rd panned output plates were subjected to standard phage ELISA, where the parallel blocking ELISA with free C225 IgG (10 μg/ml) was set to check specificity change of humanized clones Positive phage clones in phage ELISA screening were sequenced to check the diversities To express soluble Fab, g3p stump was removed from the individual phagemid vector by MIu I digestion and self-ligation Soluble Fab expression and Ni-NTA column affinity purification was performed as in-house protocol Binding evaluation of the picked Fabs were assayed in two methods: direct ELISA and BIAcore affinity analysis In direct ELISA, EGFR/Fc was coated in 1 μg/ml, samples started from 500 ng/ml, 2 times dilution down C225 IgG was used as positive control BIAcore affinity analysis was conducted in in-house facility, following standard protocol

Results

Fab Library Construction, Panning and ELISA Screening

[00299] As proof of concept, we took the mouse version of anti-EGFR Ab, M225 as our first case study sample Partial synthesized VH and VK of humanized M225 (designated as H225 in the following text) were assembled and digested separately Phagemid pMIDY was generated by site mutation in pMID21 to insert an Xho I (CTCGAG) site at 3' -terminus of VK chain H225 Fab display phage library was built by 2 steps sequential cloning of VK and VH In each Fab construct, there was a polyhistine tag (6xHis) and a Myc-tag that would facilitate detection and purification of naked Fab The final Fab library contains 5 5E9 cfu, more than 50 times more of expected total diversity Rescue and titration of phage display Fab library were performed as SOP in the lab

Panning and ELISA Screening

[00300] Huge enrichment was observed as shown by the output index after two rounds solid phase panmngs (table 14), suggesting many functional binders were present in the panned pool To isolate relatively strong binders, in the third round both standard and competition panmngs were simultaneously conducted Every setting was the same except in competition panning, high concentration (lμM) of free EGFR/Fc was added to the coated immunotube 30 minutes after phage-Fab incubation started and lasted for 3 hours before releasing bound phage We rendered that most weak binders would swing from fixed to free EGFR/Fc while strong binders could remain in complex with immobilized EGFR/Fc Indeed, as shown by the output titers, the competition panning yielded less than 1% of independent colonies as in standard panning, highlighted removal of majority of weak binders by free EGFR. Isolated colonies from 3^rd panned plates were picked for binders screening. Summary of the panning and ELISA screening was shown in table 14.

[00301] Table 6. Summary of panning M225 humanized Fab library.

[00302] As shown in table 14, output of competition panning decreased to 0.5% of standard panning in the 3^r round, indicating heavy removal of binders by free EGFR competition. Among 93 picked clones, 86 in competition panning (or 92%) in contrast to 67 (or 73%) in standard panning were positive in phage ELISA screening, in consistent with output change and further enrichment of stronger binders.

[00303] To examine the number of open-reading frame (ORF) and usage of selected residues in those diversified FR positions, Fabs DNA in 23 clones in the standard panning and 20 from competition panning were sequenced. Not surprisingly, besides every clone encoded functional Fab, the diversity was well displayed in the majority of those positions and almost every one was unique (table 14).

EXAMPLE 9 Biased Amino Acid in Diversified Spots

[00304] To find out the amino acid preference in the selected FR positions between clones from normal panning and competition panning, the amino acid sequences were compared for the selected VH (Table 7A ) and VL (Table 7B ) FR positions. After panning the library against EGFR, the amino acid preference at each of the randomized positions was not uniformly distributed, hi other words, clones that retained binding to EGFR had biases towards the amino acid substitutions at different positions of the library. For example, in VH27, VH29 and VH37, the count of one ammo acid substitution was at least four times higher than the other ammo acid choice In VH71, the frequency of R accounted for two thirds among designed options. Preference of amino- acid usage was further demonstrated in two VH positions (VH66 and VH67) Except one clone, they were exclusively limited to only one of the two available residues, implying these two positions played key roles m either supporting a functional CDR and/or stabilizing the overall domain structure Interestingly, after competed panning, while there was no big difference preferences in VH, one position in VL (L2) chose a biased amino acid (L) instead of randomly picking between L and I, although the two have similar physiochemical properties This revealed the potential importance of L2 in keeping a strong binding affinity to this Ab

[00305] Table 7 A Amino Acid statistics of sequenced humanized positive binding clones in VH Chain

Table 7B. Amino Acid statistics of sequenced humanized positive binding clones in VL Cham

Soluble Fab Evaluation

[00306] 10 clones that were derived from competed panning were expressed and purified. Although they differed in very limited positions, the yield of these clones varied drastically (Table 8 ), as large as 50 times In general, it was noted that the association (Kon) rate was well maintained among these clones, with some of them having higher values, signifying that their binding was faster compared to wild type C225 Fab. Kd are generally close to wild type except HlO and HH, which have higher dissociation rates Still, some clones (H8 and H3) possessed even lower apparent KD than C225 Fab, implying comparable or even better clones could be selected from the humanized library and no further affinity maturation was necessary for this humanization strategy to be used.

[00307] Table 8. BIAcore analysis of humanized M225 Fabs to EGFR

C225 fab was expressed in mammalian expression systems.

[00308] Binding of Fab clones to EGFR by ELISA assay was also compared (Figure 17). The same C225-Fab was used as positive control. All of the Fabs demonstrated binding to EGFR at higher concentration, eight of them showed similar binding curve as C225-Fab, two of them (HlO and HIl) had weaker binding than wild- type Fab. This data was consistent with SPR results. Both of them proved that after the humanization, the affinity of original Ab was well kept in part of the isolates.

EXAMPLE 10 Humanized sequences of positive ELISA phage screening clones

[00309] The 20 phage ELISA screening positive clones were sequenced and their amino-acids primary sequences were aligned together to indicate diversity in the previously indicated hot positions in HCFR (Figure 18A) and in LCFR (Figure 18B).

Claims

1. A process for preparing a library of affinity-optimized antibody templates, said process comprising:

I) compiling structural and sequence information regarding a large non- redundant dataset of human antibodies of known specificity and assigning: i. a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; or, ii. a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; whereby,

III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of said positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or said positive B to M score and a positive human diversity score.

2. A process for preparing a library of affinity-optimized antibody templates, said process comprising:

I) compiling structural and sequence information regarding a large non- redundant dataset of human antibodies of known specificity and assigning: i) a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity ; or, ii) a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; whereby,

II) said positive contact ratio or positive B to M score is combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; wherein said human diversity score is determined as a frequency of diversity at a Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45; or, III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of said positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or said positive B to M score and a positive human diversity score.

3. The process of claim 2, wherein said contact ratio score is determined as the ratio between the number of complementarity determining region (CDR) residues within 6 A contact of an Framework Region (FR) residue and the number of total residues within 6 A contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%.

4. The process of claim 2, wherein said B to M score is determined as the frequency of

Framework Region (FR) residue mutation which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding.

5. The process of claim 1 or 2, further comprising the step of selecting a murine monoclonal antibody of known specificity for optimization and grafting a complementarity determining region (CDR) of said murine monoclonal antibody onto said library of said human antibody templates.

6. The process of claim 5, wherein said murine antibody is M225.

7. The process of claim 5, wherein said human antibody templates comprise non-framework regions derived from a single human antibody, sharing a low sequence similarity with said murine monoclonal antibody.

8. The process of claim 5, whereby said human antibody template framework is derived from the heavy chain variable region subgroup III (VH III) and the light chain variable region Kappa III (VK III).

9. The process of claim 7, wherein said shared low sequence similarity further comprises a shared sequence identity of less than 85%.

10. The process of claim 9, wherein said similarity shared by the M225 murine antibody with a human antibody template is 69% for the heavy chain and 76% for the light chain.

11. The process of claim 1 or 2, whereby said structural information comprises X-ray crystallography data obtained from antibodies which are in complex with a known antigen and compared to crystallography data obtained from antibodies in an uncomplexed state.

12. The process of claim 1 or 2, whereby said library is a nucleic acid library or a phage display library.

13. The process of claim 1 or 2, whereby said library is an oligopeptide library.

14. The process of claim 1 or 2, wherein said process yields a Fab fragment library.

15. The process of claim 1 or 2, whereby each antibody template comprises a framework region (FR) region possessing residues having a B to M score greater than or equal to 14, and a human diversity score greater than or equal to 0.45.

16. The process of claim 1 or 2, whereby no structural modeling is required to prepare said library.

17. A library of affinity-optimized antibodies of known specificity prepared according to the process of claim 1 or 2.

18. An isolated humanized antibody selected from said library in claim 17.

19. A method of identifying a humanized antibody optimized for affinity to a known target, said method comprising the steps of:

I) compiling structural and sequence information regarding a large non- redundant dataset of human antibodies of known specificity and assigning: i. a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, ii. a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; whereby,

II) said positive contact ratio or positive B to M score is further combined with a positive human diversity score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity;

III) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score;

V) determining a respective binding affinity for a target for each of said antibodies in said library; and

VI) identifying an antibody from said library having the highest binding affinity for said target.

20. A method of identifying a humanized antibody optimized for affinity to a known target, said method comprising the steps of:

I) compiling structural and sequence information regarding a large non-redundant dataset of human antibodies of known specificity and assigning: i. a positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity, ii. a positive B to M score for particular residues in a Framework Region (FR) of said multiple antibodies of known specificity; whereby,

III) wherein said human diversity score is determined as a frequency of diversity at a Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45; or,

IV) assembling a library of human antibody templates, whereby each antibody in said library comprises an Framework Region (FR) region possessing residues having a combination of a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score;

V) grafting a complementarity determining region (CDR) of a murine antibody of desired specificity onto each antibody template in said library;

VI) determining a respective binding affinity for a target for each of said antibodies in said library formed; and

VII) identifying an antibody from said library having the highest binding affinity for said target.

21. The method of claim 19 or 20, wherein said contact ratio score is determined as the ratio between the number of complementarity determining region (CDR) residues within 6 A contact of an Framework Region (FR) residue and the number of total residues within 6 A contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%.

22. The method of claim 19 or 20, wherein said B to M score is determined as the frequency of Framework Region (FR) residue mutation which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding.

23. A humanized antibody optimized for affinity to a known target identified by the method of claim l9 or 20.

24. The method of claim 19 or 20, whereby no structural modeling is required to identify said humanized antibody.

25. A library of optimized human antibody templates for murine complementarity determining region (CDR) grafting, each antibody template in said library comprising:

I) a heavy and light chain of a human antibody, whose overall sequence shares a low sequence similarity with that of a murine antibody, wherein grafting of a complementarity determining region (CDR) of said murine antibody onto said human antibody is desired; and

II) an FR region in said human antibody possessing residues comprising a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score, whereby: i. said positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) is determined as the ratio between the number of complementarity determining region (CDR) residues within 6 A contact of an FR residue and the number of total residues within 6A contact of said FR residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%; ii. said positive B to M score for particular residues in a Framework Region (FR) is determined as the frequency of Framework Region (FR) residue mutations which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding; and iii. said positive human diversity score is determined as a frequency of diversity at an Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45.

26. The library of claim 25, further comprising a complementarity determining region (CDR) isolated from a murine antibody, which is grafted onto said Framework Region (FR) region.

27. The library of claim 25, whereby said human antibody's heavy chain's and light chain's overall sequences respectively share less than 61% identity with said murine antibody.

28. The library of claim 25, wherein said similarity shared by said murine antibody with a human antibody template is 69% for the heavy chain and 76% for the light chain.

29. An antibody isolated from the library of claim 25.

30. A Fab isolated from the library of claim 25.

31. An antibody template isolated from the library of claim 25.

32. A library of human antibody templates, wherein each antibody template in said library comprises an Framework Region (FR) region possessing residues having a combination of a positive complementarity determining region (CDR) contact ratio score or a positive B to M score and a positive human diversity score, or a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score,

I) wherein said contact ratio score is determined as the ratio between the number of complementarity determining region (CDR) residues within 6 A contact of an FR residue and the number of total residues within 6 A contact of said Framework Region (FR) residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%, said B to M score is determined as the frequency of FR residue mutation which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding and said human diversity score is determined as a frequency of diversity at an Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45.

33. An antibody template isolated from the library of claim 32.

34. A kit for preparing a library of human antibody templates, said kit comprising:

I) a library of polynucleotides encoding human antibody templates comprising Framework Region (FR) regions possessing residues comprising a positive complementarity determining region (CDR) contact ratio score or a positive B to M score and a positive human diversity score, or a positive complementarity determining region (CDR) contact ratio score and a positive human diversity score or a positive B to M score and a positive human diversity score, whereby: i. said positive complementarity determining region (CDR) contact ratio score for particular residues in a Framework Region (FR) is determined as the ratio between the number of complementarity determining region (CDR) residues within 6A contact of an Framework Region (FR) residue and the number of total residues within 6 A contact of said Framework Region (FR) residue and said positive complementarity determining region (CDR) contact ratio score is a score of greater than or equal to 25%; ii. said positive B to M score for particular residues in a Framework Region (FR) is determined as the frequency of Framework Region (FR) residue mutations which results in restoring binding of a humanized antibody to a target and said positive B to M score is a score of greater than 14 mutations at a particular Framework Region (FR) residue in an antibody, which restored said binding; and iii. said positive human diversity score is determined as a frequency of diversity at an Framework Region (FR) residue, which is present in human antibody sequences and wherein a positive human diversity score is a score of greater than 0.45; and

II) optionally a polynucleotide encoding a murine antibody fragment comprising a complementarity determining region (CDR) region, whose grafting onto said library of human antibody templates is desired and reagents for grafting said nucleic acid encoding a murine antibody fragment comprising complementarity determining region (CDR) regions onto said library of human antibody templates.

35. The kit of claim 34, wherein said kit comprises a vector comprising said polynucleotides encoding human antibody templates.

36. The kit of claim 34, wherein said kit comprises bacteriophages comprising said polynucleotides encoding human antibody templates.