WO2005021595A1 - Methods of antibody engineering using antibody display rules - Google Patents

Abstract

The present invention provides methods for modifying antibodies and antibody fragments by following certain Antibody Display Region (ADR) rules, thereby resulting in improved production of antibodies and fragments that have been modified as compared to an unmodified antibody. In one embodiment, antibodies are modified by the introduction of a heterologous peptide into one or more complementarity determining regions (CDRs). Adherence to the DR rules results in more efficient production of modified antibodies and fragments. The present invention further provides antibodies and antibody fragments modified according to the ADR rules, pharmaceutical compositions containing such modified antibodies or fragments, and therapeutic and diagnostic uses of such modified antibodies or fragments.

Description

METHODS OF ANTIBODY ENGINEERING USING ANTD3ODY DISPLAY REGION RULES

This application claims the benefit of U.S. Provisional Application Serial No. 60/499,314, filed August 28, 2003, which is incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

[0001] The present invention provides modified antibodies with new functional, structural, immunological and/or biochemical properties such that residues within Complementarity Determining Regions (CDRs) that are critical for antibody expression, structure and function are maintained. The present invention further provides methods of engineering antibodies, particularly human antibodies, according to a set of Antibody Display Region (ADR) rules to maintain or optimize antibody expression, structure, and function.

2. BACKGROUND OF THE INVENTION

[0002] Antibodies are proteins that belong to the immunoglobulin superfamily. The immunoglobulin superfamily includes T cell receptors, B cell receptors, cell-surface adhesion molecules such as the co-receptors CD4, CD8, CD19, and the invariant domains of the MHC molecules. In their soluble form, antibodies are glycoproteins produced by mature B cells, which are also called plasma cells. Antibodies are secreted into the blood and other extracellular fluids to circulate throughout the body in all animals and humans in response to foreign antigens.

[0003] Antibodies have two principal functions. The first is to recognize or bind to foreign antigens. The second is to mobilize other elements of the immune system to destroy the foreign entity. The receptors on the surfaces of immune effector cells are designed for recognition of antigens and cell surface markers on other cells. This recognition process imparts information as to whether the markers are self or non-self, and is an important element involved in modulating the immune system response to the presence of antigens. [0004] The portion of an antigen to which an antibody binds is called its antigenic determinant or epitope. Some antigens are capable of eliciting an immune response, while others are recognized as "self by the immune system. Antigens that can elicit an immune response are termed immunogens, and are usually macromolecules of at least 5000 Dalton molecular weight, such as proteins, nucleic acids, carbohydrates, and lipids. Smaller non- immunogenic molecules, termed haptens, also are capable of stimulating an immune response when coupled to a large carrier molecule.

2.1 STRUCTURE OF ANTIBODDJIS

[0005] The basic complete unit of an antibody is a four-chain Y-shaped structure.

In the early 1970s, Wu and Kabat assembled the amino acid sequences of a large collection of antibodies and demonstrated that the structure of antibodies and, in fact, all members of the immunoglobulin superfamily, consists of a constant region and four relatively conserved framework regions of semi-rigid beta sheet, with three relatively short regions of hypervariable amino acid sequence known as complementarity determining regions (CDRs or Kabat CDRs) interspersed among them (Wu and Kabat, 1970, J. Exp. Med. 132(2 :211- 250; Wu and Kabat, 1971, Proc. Natl. Acad. Sci. USA 68(7): 1501-1506). This prediction was confirmed by crystallographic studies of antibody structure (Poljak et al., 1973, Proc Natl Acad Sci USA 70(12):3305-3310; Diesenhofer et al., 1976, Hoppe Seylers Z Physiol Chem (Germany, West) 357(10):435-445; Diesenhofer et al., 1976, Hoppe Seylers Z Physiol Chem (Germany, West) 357(10): 1421-1434). CDRs may also be defined as Chothia CDRs based on the location of the structural loop regions (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

[0006] Antibodies are made up of two shorter light peptide chains linked via disulfide bonds to two longer heavy peptide chains, which are themselves connected by disulfide bonds. Each light and heavy chain of an antibody has a variable region at its amino terminus (V and V_H respectively); it is the variable region of the antibody that confers the antigen-binding specificity. A heavy chain variable domain and a light chain variable domain together form a single antigen-binding site. Thus, the basic immunoglobulin unit has two antigen-binding sites.

[0007] Diversity in the variable regions of both the light and heavy chains is restricted to the three CDRs. There are six different CDRs in each light and heavy chain pair with a total of twelve in an IgG antibody. Each CDR contains from about five to about ten amino acid residues, or up to about 20 amino acid residues (in some cases more than 20 amino acid residues) when the CDR is endogenously recombined, as is common to some antibody classes. Antibody diversity is generally created by changes in the sequences of the CDRs. Each of the three CDRs of the variable region of each light and each heavy chain forms a loop. The loops are clustered together and are connected to the four remaining parts of the variable region called the framework regions ("FRs"), which are relatively conserved among antibody molecules. Human or humanized variable region is dervied from a human antibody. In general, the human frame work region within the human heavy chain variable region is at least 50%, 60%, 70%, or 80% homologous to consensus human frame work region of a human heavy chain variable region of a human antibody. Whereas, human frame work region within the human light chain variable region is at least 65%, 70%, or 80% homologous to consensus human frame work region of a human light chain variable region of a human antibody. The variable regions are distinct for each antibody, whereas the constant regions are more highly conserved. While the light chain has only one constant region domain, the heavy chain constant region of IgG is composed of multiple domains, named CHI, CH2, and CH3. The constant region domains are charged with the various antibody effector functions, such as complement binding and binding to Fc receptors on cells such as lymphocytes, monocyte lineage cells, and other immune-effector cells. Other effector functions include differentiation, activation of the complement cell lysis system, opsonization, and attraction of macrophages. Antibodies of different isotypes have different constant domains and therefore have different effector functions. The best- studied isotypes are IgG and IgM.

[0008] All animal species express several different classes of antibodies. Five human antibody classes (IgG, IgA, IgM, IgD and IgE), and within these classes, various subclasses, are recognized on the basis of structural differences, such as the number of immunoglobulin units (i.e., heavy and light chain units) in a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. IgG antibodies are currently the most generally useful of these classes for diagnostic and therapeutic pharmaceutical uses, although antibodies from other classes may also find utility in certain uses.

2.2 ANTIBODY ENGINEERING [0009] The development of monoclonal antibody technology, first disclosed by

Kohler and Milstein (1975, Nature 256:495-497), has allowed the generation of unlimited quantities of antibodies of precise and reproducible specificity. The Kohler and Milstein procedure involves the fusion of spleen cells obtained from an immunized animal, with an immortal myeloma cell line to produce hybridomas. Clones that produce an antibody having the requisite specificity are then selected from these hybridomas. The hybridomas produce monoclonal antibodies that are uniform in their properties and specificity. [0010] To date, identification and production of suitable antibodies useful in diagnostic and therapeutic applications has depended on chance. The generation of antibody-producing hybridomas typically involves immunization of a mouse with an antigen, or, alternatively, the antigen is added to spleen cell preparations in vitro. The population of spleen cells and, therefore, of potential monoclonal antibodies with a particular specificity, depends upon the animal's immune reaction to the antigen. [0011] Additional approaches to generating antibodies useful for diagnostic and therapeutic uses have been developed as an alternative to the laborious immunization procedure mentioned above. One approach entails the cloning of antibody genes into phage, which will express on the phage surface a single variable region as described in Clackson et al, 1991, Nature 352:624; Marks et al., 1992, J. Mol. Biol. 222:581; Zebedee et al., 1992, Proc. Natl. Acad. Sci. USA 39:3175; Gram et al., 1992, Proc. Natl. Acad. Sci. USA 89:3576. Using phage library techniques, large libraries can be generated that express much of the inherent genetic diversity. However, such libraries are still constrained by the antibody repertoire from which they were derived. In yet another approach, variable domain genes that are randomly mutagenized and expressed, also result in the production of large libraries as described in Pack (1997, High Quality Antibody Libraries, Abstracts of the Eighth International Conference of Antibody Engineering). While both approaches are successful in generating great diversity, they are not much more successful in identifying useful antibodies than traditional immunization methods because they rely on random generation of CDR sequences. Moreover, antibodies generated through immunization of mice are of limited use in human therapeutics. Since mouse monoclonal antibodies are foreign and thus immunogenic to humans, they induce a human antimouse antibody (HAMA) response (Shawler et al., 1985, J. Immunol. 135:1530; Chatenaud et al., 1986, J. Immunol. 137:830).

2.3 ANTIBODY BASED PHARMACEUTICALS

[0012] The efficacy of a pharmaceutical is often derived from the ability of the pharmaceutical to enhance, antagonize or mimic the binding of one molecule to another (e.g., a ligand to its receptor, or a pathogen to a cellular receptor), thereby achieving certain physiological and pharmacological activity useful for disease prevention or amelioration. Until recently, pharmaceuticals were limited to serendipitously discovered synthetic or natural products, and were small molecule effectors that mimicked the binding of naturally occurring ligands. Even when information is available concerning the structure of ligands or their binding sites, currently available methods have not readily led to the development of effective pharmaceuticals. Methods such as the use of molecular modeling to design small molecule analogs based on crystal structure data for ligand-receptor binding pairs, or the screening for binding to a receptor using peptide combinatorial libraries or natural product extracts, have not proved to be reliable. Additionally, these synthetic or natural products do not always have the ability to discriminate in binding affinity and specificity for receptor subtypes, which can result in undesirable side effects due to insufficient control over the pharmacological effects.

[0013] There is a need for a method to more directly reproduce or inhibit the effects of natural interactions. There is a need for a method to design specific pharmaceutical agents that interact with members of a particular binding pair and which more closely mimic the behavior of naturally occurring ligands. PCT Publication WO 99/25379, entitled "Immunoglobulin Molecules Having a Synthetic Variable Region and Modified Specificity" by Burch, filed November 13, 1998, which is hereby incorporated by reference in its entirety, discloses a method for preparing antibodies that have been engineered to interact with a member of a particular binding pair by introducing into a CDR of the antibody variable domain the binding domain of the other member of the binding pair. Such engineered antibodies are termed herein SYNTHEBODY™ molecules. [0014] There is a need for improved methods to engineer antibodies, particularly human or humanized antibodies, so as to improve antibody affinity, stability and/or expression levels.

[0015] The present invention provides a method to engineer antibodies having improved affinity, stability and/or expression levels. The present invention further provides a method for engineering antibodies to contain a heterologous peptide that is a binding domain for a member of a binding pair. The present invention further provides a method for engineering humanized antibodies or other antibodies having improved characteristics for use as pharmaceutical agents.

[0016] Citation of references herein above shall not be construed as an admission that such references are prior art to the present invention.

3. SUMMARY OF THE INVENTION

[0017] The present invention is based, in part, upon the inventors' recognition that the various amino acid residues comprising the Kabat and/or Chothia CDRs (i.e., the regions of sequence and structural hypervariability, respectfully, as defined in Section 3.1 below) may not be equally modifiable (e.g., by inserting into the CDR, or by replacing all or a portion of the CDR with, a heterologous peptide). Thus, modifications at certain amino acid residues may have less of an effect on structural and functional integrity of the antibody than modifications at other amino acid residues. Specifically, the CDRs of an antibody, although defined by sequence and structural hypervariability of the variable domains of the heavy and light chains of the antibody, also contribute to antibody structure. Thus, alteration of a CDR by inserting a heterologous sequence into, or replacing all or a portion of, that CDR, may compromise antibody structure and function to a greater or lesser degree depending on where the alteration occurs. The inventors have identified amino acid residues within the CDRs, including in human antibodies, which have a greater effect on antibody structure and function. Thus, maintenance of all or some of these amino acid residues when engineering an antibody should assist in improving certain parameters of the engineered antibody, such as its structural and functional integrity, the efficiency with which it is recombinantly expressed, antigen specificity and binding affinity. In particular, the inventors, through structural and sequence analysis of CDRs of a number of antibodies, have identified conserved amino acid residues and structural motifs within the CDRs that have lower tolerance for alteration (i.e., modification of these amino acid residues has a greater impact on antibody parameters such as recombinant expression levels, antigen specificity and binding affinity), as well as amino acid residues that have a higher tolerance for modification, thereby permitting antibody engineering without significant compromise of antibody structural and functional integrity. For example, the inventors have identified amino acid residues within each CDR that can be replaced, or where individual amino acid residues or a heterologous peptide can be inserted without significantly destabilizing the antibody structure, such that the resulting antibody is efficiently expressed, the heterologous sequence, if present, is accessible to its binding partner, and the engineered antibody binds the binding partner. The regions of the CDRs comprising these amino acid residues are called "Antibody Display Regions" (ADRs) and are defined more fully herein below. The description of these ADRs sets forth basic principles and guidelines (also referred to as "ADR rules") for modifying antibodies in accordance with the instant invention. [0018] More specifically, using the sequence and structural analyses described below, the inventors have defined ADRs for each of the CDRs of human antibody heavy and light (λ and K) chains. All numbering of the amino acid residues used herein in connection with the ADR rules is according to the numbering system of Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5 Ed., U.S. Dept. Health & Human Services, Bethesda, Maryland (which is incorporated herein by reference in its entirety). Accordingly, the numbering of each amino acid residue corresponds to the number of that residue under the Kabat numbering system before any insertion or deletion is introduced to the particular ADR. Amino acid residues described as being within an ADR represent those residues that have a higher tolerance for modification than residues outside the ADR. However, when making modifications, it is not necessary to replace all the amino acid residues in a given ADR.

[0019] Accordingly, the present invention is directed to a modified antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, which modified antibody specifically binds a first member of a binding pair, which binding pair consists of a first member and a second member, said modified antibody comprising a heterologous peptide contained entirely within at least one antibody display region (ADR) of said variable region, said heterologous peptide containing a portion of said second member, which portion binds said first member, and wherein said second member is not an immunoglobulin.

[0020] With respect to the heavy chain, ADR1-H (i.e. , the ADR associated with

CDRl of the heavy chain) is defined as the region consisting of the amino acid residue from position 30 up to and including the amino acid residue at position 33 (i.e. , any of the amino acid residues from position 30 up to and including position 33 may be modified, whereas other CDR-H1 residues are preferably to be maintained). However, if the amino acid residue at position 29 is a hydrophobic residue (i.e., valine, leucine, isoleucine, phenylalanine, tryptophan, cysteine, or methionine), then ADR1-H does extend to and includes the amino acid residue at position 28. Furthermore, if the amino acid residue at position 34 of the modified sequence is hydrophobic, then ADR1-H does extend to and includes the amino acid residue at position 35. In addition, if the amino acid residues at both positions 29 and 34 of the modified sequence are hydrophobic, then ADR1-H does extend from the amino acid residue at position 28 up to and including the amino acid residue at position 35.

[0021] ADR2-H is defined as the region consisting of the amino acid residue from position 52 up to and including the amino acid residue at position 58. However, if there is a tyrosine residue at position 59, then ADR2-H extends up to and including the amino acid residue at position 62. In addition, if there is a tyrosine residue at position 59 and a hydrophobic residue at position 63 of the modified sequence, then ADR2-H consists of the amino acid residues from position 52 up to and including position 65.

[0022] ADR3-H is defined as the region consisting of the amino acid residue from position 95 up to and including the amino acid residue at position 100, but can include even additional residues inserted at the "100" position such as 100A, 100B, 100C, 100D, 100E

100F, 100G, 100H, 1001, 100J, and even to 100K or even additional residues of the inserted heterologous sequence at the "100" position, depending on the length of the CDR3-H (Note:

CDR3-H includes the amino acid residues from position 95 up to and including position 102. CDR3-H tends to be highly variable in length, and can include anywhere from 3 to 16 or more additional amino acid residues). Any salt bridge existing between the amino acid residue at position 94 and the amino acid residue at position 101 should be preserved.

Furthermore, the hydrophobic interactions between amino acid residues 100K and 102 should be preserved. If the last amino acid residue of the modified sequence before the amino acid residue at position 101 is hydrophobic, the insertion at amino acid residue 100 can be of any length. In specific embodiments, CDR3-H including the binding site is 5 to

10, 5 to 15, 5 to 20, 11 to 15, 11 to 20, 11 to 25, or 16 to 25 amino acids in length. In other embodiments, the CDR including the binding site is at least 5, 10, 15 or 20 amino acids or is more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids in length.

[0023] With respect to the kappa light chain, ADRl-κL (i. e. , the ADR in CDRl of the kappa light chain) is defined as the region consisting of the amino acid residues from position 30, up to and including position 32 of the kappa light chain. Position 29 (two amino acids after position 27) is part of the hydrophobic interior of the variable domain and needs to be a hydrophobic amino acid. If a hydrophobic residue is present at position 29 of the modified sequence, then the ADR consists of the amino acid residues from position 27 up to and including position 32. Using the Kabat numbering convention, if CDRl of the kappa light chain is longer than 11 amino acids, the additional amino acids are identified as

27 A, 27B, 27C, etc. Thus, if CDRl of the kappa light chain is 17 amino acids in length (as in Table 2), then the amino acids of the CDR are numbered 24, 25, 26, 27, 27 A, 27B, 27C,

27D, 27E, 27F, 28, 29, 30, 31, 32, 33 and 34. In this example, where CDRl is 17 amino acids in length, amino acid 27B (the position two amino acids after position 27) should be a hydrophobic amino acid and the ADR consists of amino acid residues from position 27 up to and including position 32.

[0024] ADR2- KL is defined as the region consisting of the amino acid residues from position 50 up to and including position 53 of the kappa light chain.

[0025] ADR3-κL is defined as the region consisting of the amino acid residues from position 95A up to and including position 96 of the kappa light chain. However, if a proline residue is present at position 95, then ADR-κL is defined as the region consisting of the amino acid residues from position 92 up to and including position 96.

[0026] With respect to the lambda light chain, ADRl-λL is defined as the region consisting of amino acid residues from position 25 up to and including position 27A and amino acids residues from position 29 up to and including position 32. Residue 24 is unlikely to interact with the target binding-partner and should be maintained. If an amino acid residue at position 27B of the modified sequence is present (note: there is not necessarily a residue at position 27B in lambda light chains), it preferably has a polar side chain. If there is no amino acid residue at position 27B of the modified sequence and amino acid residue 28 of the modified sequence is hydrophobic, then ADRl-λL is defined as the region consisting of amino acid residues from position 25 up to and including position 32. [0027] ADR2-λL is defined as the region consisting of amino acid residues from position 50 up to and including position 53.

[0028] ADR3-λL is defined as the region consisting of amino acid residues from position 91 up to and including position 96, where the amino acid residue at position 91 is preferably aromatic.

[0029] In some embodiments, modified antibodies are engineered by following the

ADR rules as described above so that the amino acid residues within the CDRs that are critical for antibody structure and function (i.e., those residues that are not located within the ADRs) are maintained, or in some cases only conservative substitutions are made therein. The engineering of antibodies in accordance with the methods of the present invention comprises modifying only those amino acid residues within the CDRs that tolerate modifications (e.g., deletions, replacements, or insertions). The present invention enables the engineering of antibody molecules (e.g., to confer a new or improved binding specificity on the molecule), while maintaining (or retaining acceptable levels of) or improving functional, structural, immunological, and/or biochemical properties of the molecule. A biochemical property that can be retained at an acceptable level, or maintained or improved by applying the ADR rules of the present invention, can be selected from antibody stability, antigen binding (or binding to a non-antigen binding partner when a heterologous binding domain is introduced), efficiency of recombinant expression, antibody folding, antibody assembly, and immunogenicity. For example, the ADR rules of the present invention are useful to engineer an antibody to have a binding specificity for a new non-antigen binding partner such that the resulting engineered antibody has affinity for the new non-antigen binding partner; or increased stability; or can be efficiently expressed in a recombinant system (e.g., at a commercially useful level) from eukaryotic, and preferably mammalian, cells, or a combination of said attributes.

[0030] The skilled artisan interested in successfully engineering an antibody can use the ADR rules of the present invention to achieve these ends by identifying amino acid residues in the ADRs, which residues can be modified during the engineering process, and by also identifying amino acid residues outside the ADRs, which should not be modified during the engineering process. For antibodies that have already been engineered, the skilled artisan can improve one or more aspects of that antibody by identifying amino acid residues outside the ADRs that do not conform to the ADR rules as set forth herein (see, in particular, Tables 1, 2 & 3), and substituting one or more of those amino acid residues to particular residues in conformance with the ADR rules of the present invention. For example, the residue at position 26 of the heavy chain is not a glycine then that residue may be substituted with a glycine in conformance with the ADR rules.

[0031] Accordingly, the present invention is directed to a method of engineering an antibody to specifically bind a first member of a binding pair, which binding pair consists of a first member and a second member, said antibody contains a human or humanized heavy chain and a human or humanized light chain variable region, said method comprising introducing entirely within an ADR of said antibody, a heterologous peptide comprising a portion of said second member, which portion binds said first member.

[0032] In another embodiment, the present invention is directed to a method of engineering an antibody to specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said antibody contains a human or humanized heavy chain and a human or humanized light chain variable region, said method comprising: (a) identifying an ADR in said antibody; and (b) introducing entirely within said ADR, a heterologous peptide comprising a portion of said second member, which portion binds said first member.

[0033] In one embodiment, an antibody engineered in accordance with the present invention specifically binds an antigen molecule. In another embodiment, an antibody engineered in accordance with the present invention is recombinantly expressed in cell culture, e.g., in mammalian or other eukaryotic cells (e.g., myeloma cells, CHO cells, etc.), at a sufficient level (e.g., at a level of at least 200 mg/L, at least 400 mg/L, at least 500 mg/L, at least 700 mg /L, or at least 1000 mg/L of media), and exhibits proper folding and assembly upon recombinant production such that the antibody specifically binds to its antigen or non-antigen binding partner. In one embodiment, an antibody that was previously humanized or engineered is subsequently re-engineered in accordance with the methods of the invention to improve certain attributes of the antibody. For example, an antibody is re-engineered in accordance with the methods of the present invention so as to improve the yield of the recombinantly expressed antibody to provide at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 10-fold, at least a 15-fold, or at least a 20-fold increase in yield compared to the parent antibody.

[0034] Antibodies engineered in accordance with the methods of the present invention have sufficient, and preferably therapeutically useful, avidity and/or affinity for their cognate binding partner, which can be determined using standard methods known in the art. In one embodiment, a modified antibody of the present invention binds with at least the same affinity and/or avidity, or with an enhanced affinity and/or avidity, for its cognate binding partner relative to the native binding partner, such as a ligand, and, preferably, with an affinity and/or avidity enhanced by at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold compared to the native ligand binding to the receptor. In one embodiment, antibodies of the present invention have altered kinetic parameters for the interaction with their cognate binding partner as determined using methods known to one skilled in the art. The modified antibodies of the present invention preferably bind their cognate binding partner with a K_off of less than 3 x 10^"3 s^"1, or less than 1 x 10^"3 s^"1. In another embodiment, modified antibodies of the present invention bind their cognate binding partner with a K_0ff of less than 5 x 10^"3 s^"1, less than 10^"3 s^"1, less than 8 x 10^"4 s^"1, less than 5 x 10^"4 s^"1, less than 10^"4 s^"1, less than 9 x 10^"5 s^"1, less than 5 x 10^"5 s^"1, less than 10^{" 5} s^"\ less than 5 x 10^"6 s^"1, less than 10^"6 s^"1, less than 5 x 10^"7 s^"1 or less than 10^"7 s^"1. [0035] In some embodiments, antibodies engineered in accordance with the methods of the present invention have an improved stability relative to the parent antibody, i.e., the antibody prior to modification. As used herein, the terms "stable," "stability", and the like, refer to antibodies engineered in accordance with the methods of the present invention that retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the biological or immunological activity under accelerated storage conditions relative to the engineered antibody prior to subjecting the antibody to such conditions. [0036] In another embodiment, the present invention provides improved humanization (or other form of engineering) of an antibody by altering CDR residues in accordance with the ADR rules. For example, the present invention provides a method of improving humanization of an antibody so that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17 or 20) of the original CDR residues of the acceptor human antibody (i.e., the antibody into which CDRs from a donor rodent monoclonal or other non-human antibody are grafted) that are to be conserved according to the ADR rules (i.e., those that are outside the ADR) are maintained in the humanized antibody, while one or more of the CDR residues that have a higher tolerance for modifications (i.e., those within the ADRs) are replaced with their counterpart residues from the CDRs of the non-human donor monoclonal antibody (or otherwise altered). Preferably, humanized antibodies that are engineered in accordance with the methods of the present invention have one or more improved properties as compared to antibodies made by traditional humanization procedures. [0037] In an embodiment, the present invention is directed to a method of improving a humanized or engineered antibody having a human or humanized light chain variable region and a human or humanized heavy chain variable region, and specifically binding an antigen, said method comprising making at least one amino acid substitution in at least one

CDR of said humanized or engineered antibody, wherein said at least one amino acid substitution replaces an amino acid residue that does not conform to the ADR rules with an amino acid residue that conforms to the ADR rules, wherein said improved humanized or modified antibody specifically binds said antigen.

[0038] In another embodiment, the present invention is directed to a method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region and specifically binding an antigen, said method comprising: (a) identifying at least one amino acid residue in a CDR of said humanized or engineered antibody that does not conform to the ADR rules; and (b) making an amino acid substitution at said at least one amino acid residue that does not conform to the ADR rules with an amino acid residue that conforms to the ADR rules, wherein said improved humanized or modified antibody specifically binds said antigen.

[0039] In another embodiment, the present invention is directed to a method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, and specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said humanized or engineered antibody comprising a heterologous peptide contained entirely within an ADR, said heterologous peptide containing a portion of said second member, which portion binds said first member, said method comprising making at least one amino acid substitution in at least one CDR of said humanized or engineered antibody to replace an amino acid residue that does not conform to the ADR rules with an amino acid that conforms to the ADR rules, wherein said improved humanized or modified antibody specifically binds said first member.

[0040] In another embodiment, the present invention is directed to a method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, and specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said humanized or engineered antibody comprising a heterologous peptide contained entirely within an ADR, said heterologous peptide containing a portion of said second member, which portion binds said first member, said method comprising: (a) identifying at least one amino acid residue that does not conform to the ADR rales in a CDR or said humanized or engineered antibody; and (b) making an amino acid substitution at said at least one amino acid residue that does not conform to the ADR rules with an amino acid that conforms to the ADR rules, wherein said improved humanized or modified antibody specifically binds said first member.

[0041] SYNTHEBODY™ molecules are immunoglobulins engineered to have a

CDR containing a heterologous peptide comprising a binding domain. By engineering such a SYNTHEBODY™ molecule in accordance with the ADR rules of the present invention, a

SYNTHEBODY™ molecule having improved properties can be created. For example, by introducing a heterologous peptide comprising a binding domain into a CDR in accordance with the ADR rales of the present invention, the accessibility of the heterologous binding domain to its binding partner can be optimized, and the binding affinity of the

SYNTHEBODY™ molecule for the other member of the binding pair maximized.

Different ADRs within the antibody (i.e., ADR1, 2, and 3 of each of the heavy and light chains) can promote different types of conformations of inserted peptides, e.g., loop, twisted β-strand, β-strand, and random coil. Accordingly, the ADR is preferably selected, at least in part, based upon its ability to promote the active conformation of the heterologous peptide.

Additionally, an introduction of a heterologous peptide into a CDR can often destabilize an antibody, and inhibit its recombinant production. Introduction of a heterologous peptide into a CDR in accordance with the ADR rules of the present invention can avoid these problems.

[0042] Accordingly, in some embodiments, the invention provides a modified antibody comprising a heterologous peptide that has been introduced into a CDR of the antibody in accordance with these ADR rules. In one embodiment, the heterologous peptide comprises a binding domain of a second member of a binding pair that specifically binds the first member of a binding pair. The modified antibody preferably retains structural integrity as characterized by a high level of expression, which reflects proper assembly and folding.

The modified antibody also preferably displays the binding domain of the heterologous peptide on the surface of the antibody so that the binding domain is accessible for binding to the first member of the binding pair. In another embodiment, the modified antibody is recombinantly expressed at a level that is significantly higher than that of a comparable antibody that has also been modified by the introduction of the same heterologous peptide, but in violation of the ADR rules of the present invention.

[0043] Thus, in some embodiments, the present invention provides a modified antibody having a heterologous peptide inserted into a CDR in accordance with the ADR rules of the present invention (either by insertion of the heterologous peptide into the CDR without replacing any of the existing amino acid residues of the CDR or by replacing one or more of the amino acid residues of the CDR with the heterologous peptide). In certain embodiments, the heterologous peptide is not naturally found in the CDR of an antibody. In certain embodiments, the heterologous peptide contains a binding domain from a second member of a binding pair, which binding domain can bind to a first member of the binding pair. The binding pair can be any two molecules that specifically interact with each other. In certain embodiments, the binding pair is an antigen-antibody binding pair. In other embodiments, the binding pair is not an antigen-antibody binding pair. In specific embodiments, the first member of the binding pair is a cancer antigen (i.e., a molecule expressed on the surface of a cancer cell), an antigen of an infectious disease agent (i.e., a molecule on the surface of an infectious disease agent) or a cellular receptor for an infectious disease agent. Cancer antigens include, e.g., human milk fat globule antigen (HMFG), epidermal growth factor receptor (EGFR), an epitope of polymorphic epithelial mucin antigen (PEM), and a human colon carcinoma-associated protein antigen (CCA). An antigen of an infectious disease agent includes, e.g., a Brambell receptor (FcRB), or an antigen of HS V-2, gonococcus, Treponema pallidum, Chlamydia trachomatis or human papillomaviras. In other specific embodiments, the binding pair is a receptor-ligand binding pair such as, for example, where the ligand elicits intracellular signaling by binding to its cognate receptor. In another embodiment, the binding pair is an enzyme-substrate binding pair. In one non-limiting embodiment, the binding pair is somatostatin and somatostatin receptor.

[0044] The invention further provides a method of treating or preventing a condition by using a modified antibody of the present invention. For example, antibodies having an ADR that has been modified to contain the binding site for a cancer antigen, or an antigen of an infectious agent, or a cellular receptor for an infectious disease agent, can be used to treat or prevent a cancer or an infection or disease associated with an infectious agent associated with the expression of the particular antigen. Also, for example, antibodies having an ADR that has been modified to contain the binding site for an antigen of a particular infectious disease agent, can be used to treat or prevent an infectious disease caused by the infectious disease agent. Also, for example, antibodies having an ADR that has been modified to contain either the binding site of a cellular receptor to which a ligand binds, or the cellular receptor-binding portion of a receptor ligand, can be used to treat or prevent a condition associated with overexpression or overactivity of the cellular receptor. [0045] The invention further provides methods for screening, detecting or diagnosing the presence of a condition, antigen or pathogen in a patient, or the presence of a pathogen or antigen in a biological sample, using a modified antibody of the invention. For example, antibodies modified according to the present invention to have an ADR containing the binding site for a cancer antigen, or to have an antigen of an infectious disease agent, can be used in screening, detecting or diagnosing a cancer associated with the expression of the particular cancer antigen, or an infectious disease associated with expression of the particular antigen of the infectious disease agent.

[0046] The present invention further provides therapeutic and diagnostic kits and pharmaceutical compositions containing the modified antibodies of the invention. [0047] The present invention further provides methods of producing a modified antibody of the invention.

[0048] The present invention further provides a method for preparing a pharmaceutical composition comprising admixing an effective amount of an antibody of the present invention with a pharmaceutically acceptable carrier to prepare a formulation suitable for administration to a patient.

3.1 DEFINITIONS

[0049] As used herein, the terms "antibody" and "antibodies" refer to a molecule that binds an antigen. Unless indicated otherwise, the terms refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (single-domain antibodies), single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (dsFv), intrabodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti- anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. In particular, antibodies include antibody molecules and immunologically active fragments of antibody molecules, i.e., molecules that contain an antigen binding site. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG_\, IgG₂, IgG₃, IgG , IgAt and IgA₂) or subclass.

[0050] As used herein, the terms "parent monoclonal antibody", "parent antibody" and the like refer to the monoclonal antibody into which a heterologous peptide is introduced, or which provides a constant region and framework region for a humanized monoclonal antibody.

[0051] As used herein, the term "humanized antibody" refers to an antibody in which the antigen-binding domain is derived from a non-human (e.g., murine) antibody and that otherwise contains minimal amino acid sequences derived from the non-human antibody. One kind of humanized antibody is a CDR-grafted antibody. For the most part, humanized antibodies are human antibodies (recipient antibody) in which CDRs of the recipient are replaced by CDRs from a non-human species antibody (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity, thereby transferring the binding specificity of the non-human species antibody to the human antibody. In some instances, framework region (FR) residues (the non- hypervariable or structural sequence) of the human antibody are replaced by corresponding residues from the non-human antibody. Furthermore, humanized antibodies may be engineered to comprise residues not found in the recipient antibody or in the donor antibody. Such modifications can be made to further refine or optimize characteristics, such as antigen specificity or affinity. 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 CDRs are from a non-human antibody, and all or substantially all of the FRs are those from the human antibody sequence.

[0052] For further details in humanizing antibodies, see European Patent Nos. EP

239,400, EP 592,106, and EP 519,596; International PCT Publication Nos. WO 91/09967 and WO 93/17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089,

5,766,886, and 6,407,213; Padlan, 1991, Molecular Immunology 28(4/5):489-498;

Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS

91:969-973; Tan et al., 2002, J. Immunol. 169:1119-25; Caldas et al., 2000, Protein Eng.

13:353-60; Morea et al., 2000, Methods 20:267-79; Baca et al., 1997, J. Biol. Chem.

272:10678-84; Roguska et al, 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer

Res. 55 (23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res. 55:1717-22; Sandhu, 1994,

Gene 150:409-10; Pedersen et al., 1994, J. Mol. Biol. 235:959-73; Jones et al., 1986, Nature

321:522-525; Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op.

Struct. Biol. 2:593-596, among other sources.

[0053] As used herein, the term "hypervariable region" refers to the regions of greatest sequence variability within the variable regions and generally includes amino acid residues of an antibody constituting the antigen binding site. The hypervariable regions may be "Kabat CDRs" (which include amino acid residues 24-34 (LI), 50-56 (L2) and 89-

97 (L3) in the kappa and lambda light chain variable domain, and amino acid residues 31-

35B (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain and are defined by sequence hypervariability (see Kabat et al., Sequences of Proteins of Immunological

Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), or those amino acid residues that form a structural "hypervariable loop" or

"Chothia CDRs" (which include amino acid residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain kappa and lambda variable domain and amino acid residues 26-32 (HI),

53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (see Chothia and Lesk,

1987, J. Mol. Biol. 196:901-917)).

[0054] As used herein, the terms "immunoglobulin", "immunoglobulins" and the like, refer to a ρolypeptide(s) or protein(s) expressed from an immunoglobulin gene, whether it is a wild-type, mutated, engineered, or a fragment of an immunoglobulin gene.

[0055] As used herein, the terms "modified antibody," "modified antibodies," and

"modified antibodies of the invention" refer to immunoglobulins and immunoglobulin fragments that have been engineered (i.e., one or more amino acids inserted, replaced, deleted, or combination thereof), wherein the engineering results in alteration of one or more properties of the modified antibody compared to a native antibody.

[0056] As used herein, the term "derivative" in the context of an immunoglobulin or antibody refers to an immunoglobulin or antibody that has been altered by one or more amino acid residue substitutions, deletions or additions or a combination thereof. The term

"derivative," as used herein, also refers to an immunoglobulin or antibody that has been modified by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an immunoglobulin or antibody may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or linkage to a cellular ligand or other protein. A derivative immunoglobulin or derivative antibody can be produced by chemical modification using techniques known in the art including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

Furthermore, a derivative immunoglobulin or derivative antibody preferably possesses a function (such as, e.g., antigen-binding specificity or binding affinity) that is similar or identical to that of the immunoglobulin or antibody from which it was derived.

[0057] As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agent. The use of the term "in combination" permits, but does not require, administration of the different agents combined in the same formulation, nor does it restrict the order in which prophylactic and/or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,

4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,

3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject. The prophylactic or therapeutic agents are preferably administered to a subject in a sequence and within a time interval such that a first agent of the combination can act together with the other agent of the combination to provide an increased benefit than if they were administered otherwise.

[0058] As used herein, the terms "manage," "managing", "management", and the like, refer to the beneficial effect(s) that a subject derives from administration of a prophylactic or therapeutic agent, but which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more prophylactic or therapeutic agents to "manage" a disease, condition or symptom thereof, so as to prevent or slow the progression or worsening of the disease, condition or symptom thereof.

[0059] As used herein, the terms "prevent," " preventing", "prevention", and the like, refer to preventing the onset, recurrence, or spread of a disease, condition or symptom in a subject through administration of a prophylactic or therapeutic agent.

[0060] As used herein, the term "side effects" encompasses any unwanted or adverse effects resulting from administration of a prophylactic or therapeutic agent.

Adverse effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect from a prophylactic or therapeutic agent might be harmful or uncomfortable or risky. Side effects from chemotherapy include, but are not limited to, gastrointestinal toxicity such as, e.g.,, early and late-forming diarrhea and flatulence, nausea, vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, xerostomia, as well as constipation, nerve and muscle effects, temporary or permanent damage or failure of kidneys and bladder, flu-like symptoms, fluid retention, and temporary or permanent infertility. Additional undesired effects typically experienced by patients are numerous and known in the art. Many are described in the Physicians' Desk Reference (56^th ed., 2002).

[0061] As used herein, the terms "subject" and "patient" are used interchangeably.

A subject is preferably a mammal such as, e.g., primate or non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.), more preferably a primate (e.g., monkey and human), and most preferably a human.

[0062] As used herein, the terms "treat," "treating", "treatment", and the like, refer to the eradication, reduction or amelioration of one or more symptoms of a disease or condition, such as, e.g., the eradication, removal, modification, or control of primary, regional, or metastatic cancer tissue, which results from the administration of one or more therapeutic agents or treatments. In certain embodiments, such terms refer to minimizing or delaying the spread of cancer as a result of the administration of one or more therapeutic agents to a subject with such a disease.

4. BRrøF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 Nucleotide sequence and corresponding amino acid sequence of the consensus antibody kappa light chain variable domain. The nucleotide sequence and corresponding amino acid sequence of the consensus antibody kappa light chain variable domain are shown, starting with the ATG start codon and ending 27 base pairs downstream of CDR3. The sequences and locations of the CDRs are shown.

[0064] FIG. 2 Amino acid sequences of light chain CDRl and CDRl and of nine modified constructs. The amino acid sequences of the consensus antibody kappa light chain CDRl and CDR2 as defined by Chothia and Kabat are shown as labeled. Underlined residues have been identified through the Antibody Display Rules of the present invention and are involved in interactions important for the proper folding of the variable domain. Nine modified constructs are also shown, labeled A through I, where the inserted peptide phage display derived sequence (CRFWKTWC SEQ ID NO:l) and somatostatin inner-loop sequence (KNFFWKTFTS SEQ ID NO:2) are boxed. The CDR length and change in CDR size are shown in columns to the right of the sequences.

[0065] FIG. 3. The nucleotide sequence and corresponding amino acid sequence of the consensus antibody heavy chain variable domain. The nucleotide sequence and corresponding amino acid sequence of the consensus antibody heavy chain variable domain are shown, starting with the ATG start codon and ending 32 base pairs downstream of CDR3. The modified CDR sequence is shown for Construct F.

[0066] FIG. A. Amino acid sequences of heavy chain CDRl and of one modified construct. The amino acid sequences of the consensus antibody heavy chain CDRl as defined by Kabat is shown as labeled. Underlined residues have been identified through the Antibody Display Rules of the present invention and are involved in interactions important for the proper folding of the variable domain. One modified construct is also shown, labeled J, where the inserted somatostatin inner-loop sequence (KNFFWKTFTS SEQ ID NO:2) is boxed. The CDR length and change in CDR size are shown in columns to the right of the sequences.

[0067] FIG. 5. Nucleotide and corresponding amino acid sequences of the modified

CDRs. The nucleotide and corresponding amino acid sequences of the modified CDRs for each SYNTHEBODY™ molecule construct prepared is shown. Constructs A through G were prepared in CDRl of the kappa light chain variable domain. Constructs H and I were prepared in CDR2 of the kappa light chain variable domain and construct J was prepared in CDRl of the heavy chain variable domain. The peptide display derived somatostatin mimetic and the somatostatin inner loop sequences are underlined. [0001] FIG. 6. SYNTHEBODY¹ molecule Expression Levels. Transfections into suspension CHO cells of expression vectors encoding the consensus antibody and constracts D, E, F, and G (defined in FIG. 2) were performed in duplicate. After 7 days, the media from the cultured CHO cells were assayed in triplicate at two dilutions to determine the levels of assembled antibody secreted into the media. The graph shows the relative expression levels of assembled antibody (heavy and light chain) secreted into the media. SYNTHEBODY™ molecule expression is normalized to the expression level of the consensus antibody. Error bars indicate the standard deviation from the mean. [0002] FIG. 7. SYNTHBODY Molecule Expression Levels. Transfections into suspension CHO cells of expression vectors encoding the consensus antibody and constructs I and J (see FIG. 2 and FIG. 4) were performed in duplicate. After 7 days, the media from the cultured CHO cells were assayed in triplicate at two dilutions to determine the levels of assembled antibody secreted into the media. The graph shows the relative expression levels of assembled antibody (heavy and light chain) secreted into the media. SYNTHBODY™ molecule expression is normalized to the expression level of the consensus antibody. Error bars indicate the standard deviation from the mean. [0003] FIG. 8. SYNTHBODY™ Molecule Expression Levels. Expression levels are shown normalized to the consensus antibody. The concentration of assembled SYNTHBODY™ molecule secreted from transiently transfected CHO-K1 cells was determined by sandwich ELISA and corrected for evaporation. Transfections were performed in triplicate in each of three independent experiments and expression levels were measured in triplicate at two dilutions within the linear range of the assay. The average of all data points is shown; error bars indicate standard deviation. The expression of the consensus antibody was determined to be 3.8 mg/L.

[0004] FIG. 9. Binding of synthebodies to Somatostatin Receptor 5 Membranes.

SYNTHBODY™ molecule and natural ligand SST-14 binding to Somatostatin Receptor 5 (SSTR5) membranes were examined using ¹²⁵I-[Tyr^π]-SST-14 radioligand in a competition assay. The amount of radioligand bound to the membrane after incubation with SYNTHBODY™ molecules, consensus antibody or SST-14 is shown on the y-axis in decays per minute. Data is plotted using GraphPad Prism v3.0. All data points are n=2. Error bars indicate mean and range of data.

[0068] FIG. 10. Binding of SYNTHEBODY*™ molecules to Somatostatin Receptor 5

Membranes. SYNTHEBODY™ molecules and natural ligand SST-14 binding to SSTR5 membranes was examined using ¹²⁵I-[Tyr^π]-SST-14 radioligand in a competition assay. The amount of radioligand bound to the membrane after incubation with SYNTHEBODY™ molecules, consensus antibody or SST-14 is shown on the y-axis in decays per minute. Data is plotted using GraphPad Prism v3.0. All data points are n=2. Error bars indicate mean and range of data.

5. DETAILED DESCRIPTION OF THE INVENTION [0069] The present invention provides modified antibodies, including improved humanized antibodies, SYNTHEBODY™ molecules, and other engineered antibodies, and methods of designing, engineering, improving and producing the same in accordance with a set of rales or guidelines presented herein. These "antibody display region" (ADR) rales define certain amino acid residues within each CDR that have a higher tolerance for modification. The amino acid residues in these ADRs can be modified. They are distinguished from amino acid residues within CDRs that are critical for antibody structure and function, which have a lower tolerance for modification. Modified antibodies encompassed by the present invention include but are not limited to monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (dsFv), and epitope- binding fragments of any of the above.

[0070] A modified antibody molecule according to the present invention can be derived from any type of antibody molecule including, but not limited to, antibodies, T-cell receptors, B-cell receptors, cell-surface adhesion molecules such as the co-receptors CD4, CD8, CD19, and the invariant domains of MHC molecules. In a preferred embodiment of the present invention, the antibody molecule modified according to the present invention is selected from any class of antibody, e.g., an IgG, IgE, IgM, IgD or IgA. In one embodiment, the antibody is selected from the IgG class. In addition, the antibody may be of any subclass of the particular class of antibodies. In another specific embodiment, the antibody molecule to be modified is a T-cell receptor. In a specific embodiment, a heterologous peptide inserted into an ADR of an antibody is not a T-cell epitope or B-cell epitope.

[0071] The antibody used to generate the modified antibody can be any available antibody molecule, and is preferably a monoclonal antibody or a synthetic antibody. The antibody that is modified may be a naturally occurring or previously existing antibody, or may be synthesized from known antibody consensus sequences such as the consensus sequences for the light (kappa or lambda) and heavy chain variable regions or any other antibody consensus sequence or germline (i.e., unrecombined genomic) sequences (see, e.g., the antibody consensus and germline sequences described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5^th edition, H Publication No. 91-3242, pp 2147- 2172).

[0072] As the rules presented herein are based upon analysis of human antibody variable domains, the modified antibodies of the invention are preferably human or at least have human framework regions. The present invention provides antibodies that have been modified in accordance with the ADR rales presented herein below. Thus, amino acid residues within the CDR's that are identified herein as critical for antibody structure and function are maintained (or residues at the position are substituted with a particular amino acid residue according to the rales as set forth herein) or, in some cases, only conservatively substituted. Antibodies modified in accordance with the methods of the present invention comprise changes at one or more amino acid residues within the CDRs that are identified herein as more tolerant of modification (e.g., deletions, replacements, or insertions). The present invention encompasses engineering antibodies to confer on them new functional, structural, immunological, and/or other biochemical properties. Properties that may be achieved or improved in an antibody engineered in accordance with the ADR rales of the present invention include, but are not limited to one or more of the following: improved stability; novel or increased affinity for a specific antigen or binding partner; improved display of a heterologous peptide binding domain; retained or increased efficiency of recombinant expression; retained or improved folding and assembly of the polypeptide; retained or enhanced therapeutic efficacy; and immunogenicity. In one embodiment, antibodies engineered in accordance with the present invention retain their structural integrity. In certain embodiments, antibodies engineered in accordance with the present invention are recombinantly expressed at the same or at a higher level than the parental/unmodified antibody in a mammalian or other eukaryotic cell line, (e.g., myeloma cells, CHO cells, etc.). For example, an antibody engineered in accordance with the ADR rales disclosed herein is recombinantly expressed in cell culture at a level of at least 200 mg/L, at least 400 mg/L, at least 500 mg/L, at least 700 mg/L, at least 1,000 mg/L of media and also exhibit proper folding and assembly upon recombinant production relative to the folding and assembly of the parental antibody. In certain embodiments, such antibodies are expressed at a level of at least 10 mg/L, 100 mg/L, 500 mg/L, 1 g/L or 2 g/L). [0073] In some embodiments, the present invention provides improved humanized antibodies in which all (or in certain embodiments a subset of the residues, for example, the residues in CDR1-H, CDR2-H, CDR3-H, CDRl-κL, CDR2-κL, CDR3-κL, CDRl-λL, CDR2-λL, or CDR3-λL, or a combination thereof) of the CDR residues of the antibody that have a lower tolerance for modification, as set forth by the ADR rules presented herein, are maintained, while one or more of the CDR residues that have a higher tolerance for modification, as set forth by the ADR rales presented herein, are replaced with the amino acid residues found in corresponding positions in the CDR of a non-human antibody. In one embodiment, the invention encompasses therapeutic antibodies modified using the ADR rules of the present invention, wherein the modification confers an enhanced therapeutic efficacy compared to the unmodified therapeutic antibody. In another embodiment, an improved humanized monoclonal antibody has one or more improved characteristics compared to the parent humanized monoclonal antibody selected from improved antigen binding, improved stability, or improved recombinant production. In an antibody molecule engineered to contain a heterologous peptide (so as to provide a binding domain of a second member of a binding pair that can bind a first member of the binding pair), the invention provides the improvement comprising inserting the heterologous peptide into an ADR such that the active conformation or binding affinity, stability or recombinant expression of the antibody is retained or improved.

[0074] In some embodiments, the invention provides modified antibodies comprising a heterologous peptide comprising a binding domain of a second member of a binding pair that specifically binds to the first member of a binding pair, wherein the heterologous peptide has been introduced into an ADR of the antibody. The modified antibody preferably retains the structural integrity and, also preferably, displays the binding domain of the heterologous peptide on the surface of the antibody such that the binding domain is accessible for binding to the first member of the binding pair. In such an antibody, the heterologous peptide is accessible to solvent (and the other member of the binding pair) and adopts a bioactive conformation, e.g., it binds specifically to the other member of the binding pair. [0075] The present invention further provides a method of treatment using a modified antibody of the invention. For example, antibodies having an ADR modified to contain the binding site for a cancer antigen can be used to treat the cancer. Also for example, antibodies having an ADR modified to contain a cellular receptor or receptor ligand for an infectious disease agent can be used to treat a disease or condition associated with the infectious disease.

[0076] The invention further provides methods for screening for, or diagnosing a cancer or infectious disease using the modified antibodies of the invention.

5.1 MODIFIED ANTIBODIES

[0077] The present invention encompasses modified antibodies wherein one or more

ADRs within one or more CDRs have been modified according to the ADR rules disclosed herein. The ADRs are defined herein as regions in each CDR that have a higher tolerance for amino acid insertions, deletions, and/or substitutions such that these regions can be so modified while minimizing the loss of structural integrity and the loss of function, such as binding activity of the antibody. The ADRs have been defined herein by comparative sequence analysis so as to identify: (i) amino acid residues in each CDR that are highly conserved and, thus, are likely to play a critical role in antibody structure and function; and (ii) amino acid residues in each CDR that may be varied, e.g., by insertions, deletions or substitutions. Specifically, using the sequence and structural analysis described below, ADRs for each of the CDRs of human antibody heavy and light (λ and K) chains have now been defined. All numbering of CDR residues is according to Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5^th edition, United States Department of Health and Human Services, Bethesda, Maryland, which is incorporated by reference herein in its entirety.

5.1.1 HEAVY CHAIN ADRS

[0078] With respect to the heavy chain, ADR1-H (of CDR1-H) is defined as including amino acid residue 30 up to and including amino acid residue 33. However, if amino acid residue 34 is hydrophobic, then ADR1-H does extend up to and includes amino acid residue 35, and if amino acid residue 29 is hydrophobic, then ADR1-H does extend up to and includes amino acid residue 28.

[0079] ADR2-H is defined as amino acid residue 52 up to and including amino acid residue 58. However, if amino acid residue 59 is a tyrosine residue, then ADR2-H is amino acid residue 52 up to and including amino acid residue 62. Furthermore, if there is a tyrosine residue at amino acid 59 and a hydrophobic amino acid residue at position 63, then ADR2-H is defined as amino acid residue 52 up to and including amino acid 65. A hydrophobic amino acid residue such as isoleucine is preferably located at position 51, and a tyrosine residue is preferably located at position 59.

[0080] ADR3-H is defined as amino acid residue 95 up to and including amino acid

100, but may include 100K or even additional residues at the "100" position depending on the length of CDR3 (CDR3-H includes residues 95-102, and is of variable lengths, including anywhere from 3 to 16 or even more amino acids). Any salt bridge existing between amino acid residue 104 and amino acid residue 101 should be preserved; and the hydrophobic interactions between amino acid residues 100K and 102 should be preserved. If the last residue before residue 101 is hydrophobic, the insertion at residue 100 can be of any length. In specific embodiments, the CDR including the binding site is 5 to 10, 5 to 15, 5 to 20, 11 to 15, 11 to 20, 11 to 25, or 16 to 25 amino acids in length. In other embodiments, the CDR including the binding site is at least 5, 10, 15 or 20 amino acids or is more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids in length.

5.1.2 LIGHT CHAIN ADRS

[0081] With respect to the kappa light chain, ADRl-κL (i.e., the ADR in CDRl of the kappa light chain) is defined as amino acid residue 30 up to and including amino acid residue 32 of the kappa light chain. However, if a hydrophobic residue (i.e., valine, leucine, isoleucine, phenylalanine, tryptophan, cysteine, or methionine) is present at position 29 or, if the CDR is longer than 11 amino acids, at the position that is two amino acid residues after position 27, then the ADR is amino acid residues 27 up to and including 32. Any modification of ADRl-κL should maintain a critical serine residue at position 26 and a critical hydrophobic residue at position 33.

[0082] ADR2-κL is defined as amino acid residue 50 up to and including amino acid residue 53 of the kappa light chain.

[0083] ADR3-κL is defined as amino acid residue 95A and amino acid residue 96 of the kappa light chain. However, if a proline residue is present at position 95, then ADR3-κL is defined as amino acid residue 92 up to and including amino acid residue 96. A threonine residue (or other amino acid residue having a hydroxyl-containing side chain) at position 97 is also preferably maintained, as are glutamine residues at positions 89 and 90, because these residues form hydrogen bonds with other residues within the variable domain. [0084] With respect to the lambda light chain, ADRl-λL is defined as amino acid residue 25 up to and including amino acid residue 27A and amino acid residue 29 up to and including amino acid residue 32. Amino acid residue 24 is unlikely to interact with a target and is preferably maintained unchanged from its original form. If amino acid residue 27B is present it preferably has a polar side chain. If amino acid residue 27B is missing and amino acid residue 28 is hydrophobic, then ADRl-λL is defined as including amino acid residue 25 to up to and including amino acid residue 32.

[0085] ADR2-λL is defined as including amino acid residue 50 up to and including amino acid residue 53.

[0086] ADR3-λL is defined as including amino acid residue 91 up to and including amino acid residue 96.

[0087] Tables 1, 2 and 3 below list for each of the human heavy chain, human kappa light chain, and human lambda light chain, respectively, for each position in the CDRs, all amino acid residues found in the available 3-dimensional structures along with their respective frequencies of occurrence as determined by analysis of the human antibody sequence database and the rales for modification of the particular residue. Amino acid and percent occurrence are not provided in the tables for certain positions where the Kabat numbering system does not correlate with the 3-dimensional stractures. The particular CDR amino acid residues cited in Tables 1, 2 and 3 are numbered based on the numbering system of Kabat et al., 1991 Sequences of Proteins of Immunological Interest, 5^th edition, United States Department of Health and Human Services, Bethesda, Maryland, of the human heavy, light ( ) and light (λ) chains, respectively.

Table 1: Human Heavy Chain CDR l

CDR 2

CDR3

Table 2: Human Light Chain - Kappa CDRl

CDR 2

CDR 3

Table 3: Human Light Chain - Lambda CDRl

CDR 2

CDR 3

[0088] The invention encompasses modified antibodies that are engineered according to the ADR rules presented herein so that the amino acid residues within the CDRs that are important for antibody structure and function are maintained (or, in certain embodiments, substituted with the most common residue found at that position), or in some cases only conservative substitutions thereof are made. Engineering antibodies in accordance with the ADR rales of the present invention comprises modifying those amino acid residues (and, preferably only those residues) within the CDRs that are identified herein as having a higher tolerance for modifications (e.g., deletions, replacements, and/or insertions). The present invention encompasses engineering antibodies to confer new functional, structural, immunological, and/or biochemical properties while retaining sufficient levels of expression; maintaining, improving, or optimizing stability, antigen binding, efficiency of recombinant expression, folding and assembly, immunogenicity, or some combination thereof. In one embodiment, antibodies engineered in accordance with the present invention retain their structural integrity .

[0089] In one embodiment, antibodies engineered in accordance with the methods of the present invention do not have modifications at any of positions 26, 33, 89, 90, 91, or 97 of the variable kappa light chain.

[0090] In another embodiment, antibodies engineered in accordance with the methods of the present invention do not have modifications at position 24 or 28 of the variable lambda light chain.

[0091] In another embodiment, antibodies engineered in accordance with the methods of the present invention do not have modifications at any of positions 29, 34, 59,

100K, 101, or 102 of the variable heavy chain.

[0092] In some embodiments, antibodies modified in accordance with the methods of the present invention have enhanced affinity and/or avidity for their cognate binding partner relative to the parental antibody (i.e., the antibody prior to such modification), as determined using standard methods known in the art. In a specific embodiment, modified antibodies of the present invention have an affinity and/or avidity for their cognate binding partner that is at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold higher than that of the parental antibody.

[0093] In some embodiments, the kinetic parameters for the interaction of antibodies with their cognate binding partners is determined using methods known in the art. The modified antibodies of the invention preferably bind their cognate binding partners with a

K_off of less than 3 x 10^" s^" , and, more preferably, with a K_0ff of less than 1 x 10 s . In other embodiments, modified antibodies of the present invention bind their cognate binding partner with a K_0ff of less than 5 x 10^"3 s^"1, less than 10^"3 s^"1, less than 8 x 10^"4 s^"\ less than 5 x 10^"4 s^"1, less than 10^"4 s , less than 9 x 10^"5 s^"1, less than 5 x 10^"5 s^'1, less than 10^"5 s^"\ less than 5 x 10^"6 s^"1, less than 10^"6 s^"1, less than 5 x 10^"7 s^"1, or less than 10^"7 s^"1.

[0094] In some embodiments, antibodies engineered in accordance with the methods of the invention have an improved stability relative to the parental antibody, i.e., the antibody prior to modification. As used herein the terms "stable," "stability" and the like refer to antibodies that retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of at least one biological activity, such as antigen binding, or of the structural integrity of the molecule (e.g., as evidenced by lack of degradation or lack of aggregation, etc.) under given manufacture, preparation, transportation and storage conditions relative to the antibody prior to modification. The stability of antibodies or fragments thereof engineered in accordance with the methods of the present invention can be assessed by comparing degrees of aggregation, degradation or fragmentation using methods known in the art. These include, but are not limited to, reduced capillary gel electrophoresis (rCGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size exclusion chromatography (HPSEC), compared to an appropriate reference. The overall stability of an antibody or an antigen-binding fragment can also be assessed by various immunological assays including, for example, ELISA and radioimmunoassay. [0095] In one embodiment, formulations of antibodies of the present invention preferably exhibit low to undetectable levels of aggregation as measured, e.g., by HPSEC or rCGE, that is, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, and most preferably no more than 0.5% aggregate by weight protein. In another embodiment, formulations of antibodies of the present invention preferably exhibit low to undetectable levels of fragmentation, i.e., intact antibodies represent 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 99.5% or higher of the total weight of protein after storage, e.g., after storage for 30 min, 1 hr, 5 hrs, 1 day, 1 week, 1 month, 2 months, or 3 months, at 40°C, room temperature, or 4°C. By using SDS-PAGE, the density or radioactivity of each protein band (e.g., stained or labeled with radioisotope) can be measured. From this, the amount of non-degraded antibody can be estimated.

[0096] In another embodiment, antibodies engineered in accordance with the present invention are recombinantly expressed at sufficient levels in mammalian or enkaryotic cells for the desired use, e.g., myeloma cells such as NSO cells, CHO cells, etc., to produce 200 mg/L, at least 400 mg/L of media; at least 700 mg/L of media; or at least 1000 mg L of media, and exhibit proper folding and assembly upon recombinant production relative to the parental antibody. In other embodiments, the amount of antibodies engineered in accordance with the present invention is at least 5 g/L, 10 g/L, 15 g/L, 50 g/L, 100 g/L, of media. In one embodiment, antibodies engineered in accordance with the present invention are recombinantly expressed at a higher level relative to the parental antibody. The engineering of antibodies in accordance with the present invention may result in a significant improvement in yield of recombinantly expressed antibody such as, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in the yield as compared to the yield of the parental antibody.

[0097] In some embodiments, improved humanized antibodies have also been modified so that at least one amino acid in the acceptor antibody CDR has been replaced with the corresponding residue from the non-human donor antibody. Recipient CDRs that may be modified in accordance with the methods of the present invention include light chain CDRs (e.g., CDR-1L, CDR-2L, CDR-3L), heavy chain CDRs (e.g., CDR-1H, CDR-

2H, CDR-3H) and combinations thereof.

[0098] Humanized antibodies improved in accordance with the ADR rales of the invention should retain at least the same affinity as an antibody that has not been improved

(or the parent humanized monoclonal antibody) using the ADR rales of the present invention. These affinity levels may vary from at least about 10⁸ M^"1 to higher, and may be within 4-fold, preferably within 2-fold, of the affinity of the donor antibody for its cognate antigen. In one embodiment, antibodies modified in accordance with the ADR rales of the present invention will have affinities at least 60%, at least 70%, at least 80%, or at least

90% of the original donor (i.e., non-human) antibody's affinity for its antigen. In some embodiments, humanized antibodies modified according to the ADR rules of the present invention produce a reduced HAMA response in comparison to humanized antibodies prepared by traditional methods. Methods of antibody humanization in accordance with the present invention can be superior to methods traditionally used, i.e., humanized antibodies of the invention have minimal immunogenicity when administered to humans while maintaining antigen binding similar to that of the donor antibody.

[0099] It will be appreciated by those skilled in the art that the present invention can be widely applied to the process of humanizing antibodies by CDR-grafting. The donor

(i.e., source of CDR sequences) antibodies and acceptor (i.e., source of non-CDR sequences) antibodies can be derived from animals of the same species and even the same antibody class or sub-class. Preferably, the donor and acceptor antibodies are derived from animals of different species. Typically the donor antibody is a non-human antibody, e.g., rodent monoclonal antibody, and the acceptor antibody is a human antibody.

[00100] Improving humanized antibodies in accordance with the ADR rules of the present invention involves analysis of the sequence and stractural data and review of the information presented in Tables 1, 2, and 3, supra to select the particular amino acid residues within the recipient antibody CDRs that can be replaced with the corresponding residue of the donor antibody CDRs. Specifically, in accordance with the present invention, specific amino acid residues within CDRs have been identified that are critical for antibody structure and function because they are involved in stabilizing the tertiary and/or quaternary fold of the antibody, e.g., certain amino acid positions have numerous contacts in the hydrophobic core of the antibody structure. Similarly, specific amino acid residues within

CDRs have been identified that are mainly solvent-exposed and do not make significant contacts with other residues in maintaining the tertiary and or quaternary structure of the antibody. These residues may generally be replaced with the corresponding amino acid from a donor antibody without substantial impact on the stability or function of the antibody. For example, residues within heavy chain CDRl of the antibody that may be replaced with the corresponding residue from the CDRl of the donor antibody are at positions 30, 31, 32, and 33. Stractural analysis has indicated that amino acid residues at these positions are mainly solvent-exposed. Accordingly, they do not provide any significant interactions for maintaining the overall fold of the antibody and, thus, one or more of them may be modified. Furthermore, if the amino acid residue at position 29 is hydrophobic, then the amino acid residue at position 28 can be replaced, and if the amino acid residue at position 34 is hydrophobic, then the amino acid residue at position 35 can be replaced.

[00101] Amino acid residues within heavy chain CDRl that cannot be replaced are at positions 26 and 27. Amino acids residues at these positions appear to provide critical interactions, for example, with the hydrophobic interior of the antibody, and thus need to be maintained in order to preserve the structural and functional integrity of the antibody.

[00102] Amino acid residues within heavy chain CDR2 of the recipient antibody that can be replaced with the corresponding amino acid residue of the donor antibody CDR2 are at positions 52, 52A, 52B, 52C, 53, 54, 55, 56, 57 and 58. Amino acid residues within heavy chain CDR2 of the recipient antibody that cannot be replaced with the corresponding residue of the donor antibody CDR2 are at positions 50 and 51.

[0100] Amino acid residues within CDR3 of the heavy chain of the recipient antibody that can be replaced with the corresponding amino acid residue of the donor CDR3 are at positions 95-100J, and 100K. These residues have hydrophobic interactions with residues in the light chain. Position 101 in CDR3 of the heavy chain of the antibody must maintain a salt bridge to the basic position at position 94, and thus has to remain acidic.

Positions 101 and 102 of CDR3 of the heavy chain of the antibody should be maintained.

[0101] Amino acid residues within the kappa light chain CDRl of the recipient antibody that can be replaced with the corresponding amino acid residues in CDRl of the donor antibody are at positions 27, 28, 30, 31, 32, and 34. Amino acid residues within the light chain CDRl of the antibody that cannot be replaced with the corresponding amino acid residue of CDRl of the donor monoclonal antibody light chain are at positions 24, 25, 26,

29 and 33. Position 29 can be replaced if the CDR is longer than 11 amino acids as long as the amino acid residue that is two positions after position 27 is hydrophobic. Position 26 of the light chain CDRl is preferably a serine.

[0102] Amino acid residues within the CDR2 of the kappa light chain of the antibody that can be replaced with the corresponding amino acid residue of the donor antibody of

CDR2 are at positions 50, 51, 52 and 53.

[0103] Amino acid residues within the CDR3 of the kappa light chain of the antibody that can be replaced with the corresponding amino acid residue of the donor antibody of

CDR3 are at positions 95 A, 95B, and 96. Position 95 of the CDR3 of the kappa light chain cannot be replaced or substituted and should be maintained as a proline residue. Amino acids at positions 89-91, and 97 of the light chain CDR3 need to be maintained as H-bond donor amino acids, i.e., amino acids that can participate in H-bond formation with another amino acid in the antibody.

[0104] In addition, amino acids at positions identified as those to be maintained may be substituted with a conservative amino acid substitution, or if that residue is not the prevalent amino acid at that position, substituted with the prevalent amino acid at that postion as set forth in Tables 1, 2, and 3, supra.

[0105] The improved humanized antibody can be selected from any class of antibodies, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGi, IgG₂,

IgG₃ and IgG . Usually the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically

IgGi. Where such cytotoxic activity is not desirable, the constant domain may be of the

IgG class. The humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.

[0106] In some embodiments, the antibodies humanized in accordance with the ADR rales of the present invention further comprise framework regions substituted with the corresponding amino acid residue from a CDR donor antibody to alter, and preferably improve, antigen binding. These framework substitutions are identified by methods known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No.

5,585,089 to Queen et al.;U.S. Patent No. 6,548,640 to Winter; and Riechmann et al., 1988,

Nature 332:323, which are all incorporated herein by reference in their entireties.) [0107] The antibodies prepared according to the ADR rales of the present invention can, in turn, be utilized to generate anti-idiotype antibodies using techniques known to those skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J. 7:437-444; and Nissinoff,

1991, J. Immunol. 147:2429-2438).

[0108] The present invention further provides polynucleotide molecules comprising a nucleotide sequence encoding an antibody of the invention or a fragment thereof, and the uses thereof.

[0109] Antibodies that have been humanized in accordance with the methods of the present invention are preferably modified so that the human antibody CDR residues that are conserved residues and/or participate in conserved stractures within the CDRs, as identified herein, are maintained, while those CDR residues that can tolerate modifications are replaced with their counterpart amino acid residues from non-human species.

[0110] In some embodiments, a humanized antibody is a "derivative" further comprising one or more amino acid residue substitutions, deletions or additions in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, better binding, or worse binding to its cognate antigen when compared to the corresponding non-derivatized humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of one or more CDRs have been substituted or deleted, or one, two, three, four or five amino acid residues of one or more CDRs have been added, or some combination thereof.

[0111] In some embodiments, the present invention provides for modified antibodies having at least one ADR that contains a heterologous peptide comprising a binding site for a first member of a binding pair, i. e. , a portion of the amino acid sequence of the second member of the binding pair, such that the modified antibody specifically binds (as determined, e.g., by any method known in the art) to the first member of the binding pair.

In one embodiment, the amino acid sequence of the binding site is not found naturally within the CDR containing the ADR, and preferably not found in any CDR of any antibody.

In one embodiment, the binding pair is not an antibody-antigen binding pair. In one embodiment, the binding pair is a ligand-receptor binding pair.

[0112] Modified antibodies of the invention comprising a heterologous sequence

(e.g., a peptide comprising the binding site of one member of a binding pair) (a/k/a a

"SYNTHEBODY™ molecules") preferably maintain amino acid residues and structural elements of CDRs critical for antibody structure and function such that the structural integrity of the antibody is maintained and the peptide can adopt a biologically active conformation. In addition, by maintaining the structural integrity of the antibody, the modified antibody can be efficiently expressed recombinantly at an acceptable level for research purposes, or for therapeutic or diagnostic uses. In one embodiment, the modified antibody is expressed at a level that is at least at 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of expression of the parent antibody prior to modification according to the present invention. In one embodiment, the expression of the modified antibody in a recombinant mammalian expression system is at least 0.4 mg/ml, at least 0.7 mg/ml, and at least 1.0 mg ml.

[0113] Sequence and structural analysis have also determined the structural motif that the introduced heterologous peptide will most likely adopt. Accordingly, by determining (at least roughly) the secondary structure of the binding domain, the heterologous peptide can be introduced into the ADR that best promotes the formation of that secondary structure. Generally, all the ADRs promote the formation of loops. More specifically, ADR2-κL promotes the formation of a β-turn; ADR2-H promotes the formation of a β-strand and a random coil. Thus, an ADR can often be chosen to optimize the bioactive conformation of the heterologous peptide.

[0114] The amino acid sequence of the binding site may be identified by any method known in the art. For example, the amino acid sequence of a second member of a binding pair that is known to be directly involved in binding the first member of the binding pair can be introduced into the ADR of an antibody so that the resulting antibody specifically recognizes the first member of the binding pair. If the amino acid sequence for the binding site in the second member of the binding pair is not known, it can be determined by any method known in the art such as by, for example, molecular modeling methods or empirical methods, e.g., by assaying portions (e.g., peptides) of the second member for binding to the first member, or by making mutations in the amino acid sequence of the second member and determining which mutations reduce or prevent binding.

[0115] The second member from which the binding site is derived is a protein, while the first member may be any molecule that interacts with the second member, such as a protein, nucleic acid, carbohydrate, or lipid. In certain embodiments, the modified antibody contains a binding sequence for a cancer antigen, or an infectious disease antigen, or a cellular receptor for a pathogen, or a receptor or ligand that participates in a receptor-ligand binding pair (e.g., that elicits cell signaling).

[0116] In a specific embodiment, the binding pair is a protein-protein interaction pair, which is either a homotypic interaction (i.e., the interaction is between two of the same protein molecules), or a heterotypic interaction (i.e., the interaction is between two different protein molecules). [0117] In a specific embodiment, the first member of the binding pair is a member of a ligand-receptor binding pair, preferably of a receptor-ligand binding pair in which the ligand binds to the receptor and thereby elicits a physiological response (such as, e.g., intracellular signaling). By way of example, and not by way of limitation, the ligand or receptor can be a hormone, autocoid, growth factor, cytokine or neurotransmitter, or receptor for a hormone, autocoid, growth factor, cytokine, or neurotransmitter, or any receptor or ligand involved in signal transduction, preferably where the receptor is not an antibody. For reviews of signal transduction pathways, see, e.g., Campbell, 1997, J. Pediat. 131.S42-S44; Hamilton, 1997, /. Leukoc. Biol. 62:145-155; Soede-Bobok & Touw, 1997, J. Mol. Med. 75:470-477; Heldin, 1995, Cell 80:213-223; Kishimoto et al., 1994, Cell 76:253- 262; Miyajima et al., 1992, Annu. Rev. Immunol. 10:295-331; and Cantley et al., 1991, Cell 64:281-302. In specific embodiments, one member of the binding pair is a growth enhancing factor such as, but not limited to, fibroblast growth factor, endothelial mitogenic growth factors, and epidermal growth factors, transforming growth factor, platelet derived endothelial growth factor, platelet derived growth factor, tumor necrosis factor, hepatocyte growth factor and insulin like growth factor, transcription factors, proteinkinases, thymidine kinase, and bone morphogenic proteins. In specific embodiments, one member of the binding pair is a ligand such as, but not limited to, cholecystokinin, galanin, IL-1, IL-2, IL- 4, IL-5, IL-6, IL-11, a chemokine, leptin, a protease, neuropeptide Y, neurokinin-1, neurokinin-2, neurokinin-3, bombesin, gastrin, corticotropin releasing hormone, endothelin, melatonin, somatostatin, vasoactive intestinal peptide, epidermal growth factor, tumor necrosis factor, dopamine, endothelin, endogenous opioids, glutamate, aspartate, or a receptor for any of these ligands. In other embodiments, one member of the binding pair is a receptor such as, but not limited to, opioid receptor, glucose transporter, glutamate receptor, orphanin receptor, erythropoietin receptor, insulin receptor, tyrosine kinase (TK)- receptor, KIT stem cell factor receptor, nerve growth factor receptor, insulin-like growth factor receptor, granulocyte-colony stimulating factor receptor, somatotropin receptor, glial- derived neurotrophic factor receptor or gp39 receptor, G-protein receptor class, α-adrenergic receptor, B2-adrenergic receptor, ligand-gated ion channel receptor, or a ligand that binds any of these receptors. Specific non-limiting examples of opioid receptors include μ, δ, and K type opioid receptors. In another embodiment, the binding pair is a ligand and a ligand- gated ion channel. The ligand-gated ion channel may be selected from a calcium channel, a sodium channel, or a potassium channel, among others. In certain embodiments, the present invention provides modified antibodies that can specifically bind to a receptor, and which are antagonists of the native ligand that otherwise would bind to that receptor. For example, the modified antibody can function as an antagonist of endorphin, enkephalin or nociceptin. h other embodiments, the present invention provides modified antibodies that can specifically bind to a receptor and are agonists of that receptor. For example, the modified antibody can be an agonist of the endorphin, enkephalin, or nociceptin receptor. In one embodiment, the modified antibody does not bind the fibronectin receptor. In another preferred embodiment, the binding sequence of the antibody is not Arg-Gly-Asp, is not a multimer of a binding sequence inserted into one or more CDR according to the ADR rales, and preferably is not a multimer of the sequence Arg-Gly-Asp, or is not an integrin binding site, and more preferably, is not a binding site for the β-subunit of integrin.

[0118] In other specific embodiments, an antibody is modified according to the present invention so that an ADR in the antibody contains a binding site from a transcription factor. In one aspect, the modified antibody does not bind to a specific DNA sequence, particularly, does not bind to a transcription factor binding site.

[0119] In certain embodiments, an antibody has at least one ADR that has been modified to contain an amino acid sequence of a binding site for a cancer or tumor antigen

(e.g., as described in detail in section 5.3.1, infra.). For example, an antibody has been modified so that at least one ADR contains an amino acid sequence of a binding site for an antigen of a tumor of the breast, ovary, uterus, prostate, bladder, lung, skin, pancreas, colon, gastrointestinal tract, B lymphocyte, or T lymphocyte. In one embodiment, the antigen is a human colon carcinoma-associated antigen or an epithelial mucin antigen. In another embodiment, the antigen is Epidermal Growth Factor Receptor ("EGFR"). In other embodiments, at least one ADR of an antibody is modified to contain an amino acid sequence for a binding site for a human milk fat globule receptor.

[0120] In other embodiments, at least one ADR of an antibody has been modified to contain an amino acid sequence for a binding site for an antigen of an infectious disease agent (e.g., as described in detail in section 5.4.2, infra.), or a binding site for a cellular receptor of an infectious disease agent, preferably where the binding site is not an amino acid sequence of a Plasmodium antigen, or is not the binding site Asn-Ala-Asn-Pro or Asn-

Val-Asp-Pro (or a multimer thereof). In additional embodiments, an antibody has an ADR that has been modified to contain a binding site for a bacterial or viral enzyme.

[0121] As noted supra, the present invention describes six ADRs, three on the light chain and three on the heavy chain, which are contained within the antibody CDRs. Five of these CDRs are germline CDRs (i.e., are directly derived from the germline genomic sequence of the animal, without any recombination) and one of the CDRs is a non-germline

CDR (i.e., differs in sequence from the germline genomic sequence of the animal and is generated by recombination of the germline sequences). Whether a CDR is a germline or non-germline sequence can be determined by sequencing the CDR and then comparing the sequence with known germline sequences, e.g., as listed in Kabat et al. (1991, Sequences of Proteins of Immunological Interest, 5^th edition, NIH Publication No. 91-3242, pp 2147- 2172). Significant variation from the known germline sequences indicates that the CDR is a non-germline CDR.

[0122] Accordingly, in other embodiments of the invention, the modified ADR is in a germline CDR or, alternatively, is in a non-germline CDR, or in both germline and non- germline CDRs.

[0123] A heterologous peptide can be introduced into any of the ADRs of the antibody, and it is within the skill in the art in view of this disclosure to insert the heterologous peptide into different ADRs of the antibody and then screen for recombinant expression of the modified antibody and/or the ability of the modified antibody to bind to the particular member of the binding pair, e.g., as discussed in Section 5.3, infra. Thus, one can determine the optimal placement of the heterologous peptide in any particular ADR. In specific embodiments, an ADR of either the heavy or light chain variable region is modified to contain the amino acid sequence of the heterologous peptide. In another embodiment more than one ADR contains an amino acid sequence of a heterologous peptide, or each of more than one ADR contains different heterologous peptides. In particular, embodiments, two, three, four, five or six ADRs have been modified to containa heterologous peptide. The heterologous peptides in each of the ADRs may be the same or different. In a preferred embodiment, one or more ADRs contain a binding site for a first member of a binding pair. In addition, one or more other ADRs contain a binding site for a molecule on the surface of an immune cell such as, but not limited to, a T-cell, B-cell, NK-cell, K-cell, TIL-cell or neutrophil. For example, an antibody modified to have both (i) a binding site for a cancer antigen or an infectious disease antigen and (ii) a binding site for a molecule on the surface of an immune cell, can be used to target the immune cell to either a cancer cell bearing the cancer antigen or to the infectious disease agent.

[0124] In specific embodiments of the present invention, the heterologous peptide amino acid sequence is inserted into the ADR either without replacing any of the amino acid sequence of the ADR itself or, alternatively, by replacing all or a portion of the amino acid sequence of the ADR. In specific embodiments, the amino acid sequence of the heterologous peptide replaces 1, 2, 5, 8, 10, 11, or 15 amino acids of the ADR sequence. [0125] The amino acid sequence of the heterologous peptide can be the minimal binding site necessary for the binding together of the two members of the binding pair (which can be determined empirically by any method known in the art); alternatively, the amino acid sequence of the heterologous peptide can be bigger than the minimal binding site necessary for the binding together of the two members of the binding pair. In particular embodiments, the heterologous peptide amino acid sequence is at least 4 amino acids in length, or is at least 6, 8, 10, 15, or 20 amino acids in length. In other embodiments, the amino acid sequence of the heterologous peptide is no more than 10, 15, 20, or 25 amino acids in length, or is 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20 or 10 to 25 amino acids in length. In other embodiments, the heterologous peptide is 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, or 61 to 70 amino acids in length. In other embodiments, the heterologous peptide is greater than 30, 40, 50, 60, 70 or 80 amino acids in length. [0126] The total length of the ADR, or the CDR containing the ADR (/. e. , the combined length of the binding site sequence and the rest of the ADR or CDR sequence), should be of an appropriate number of amino acids to allow binding of the antibody to the binding pair member. CDRs have been observed to have a range of numbers of amino acid residues, and the observed size ranges for the CDRs are provided in Table 4 below.

Table 4 CDR Number of residues LI 10-17 L2 7 L3 7-11 HI 5-7 H2 9-12 H3 2-25 (compiled from data in Kabat and Wu, 1971, Ann. NY Acad. Sci. 190:382-93: for review see Panen, 1992, Hum. Antibodies Hybridomas 3(3): 137-45.)

While many CDR H3 regions are of 5-9 residues in length, certain CDR H3 regions have been observed that are much longer. In particular, a number of anti- viral antibodies have heavy chain CDR H3 regions of 17-24 residues in length.

[0127] Accordingly, in specific embodiments of the present invention, the CDR which contains the ADR having the binding site is within the size range provided for that particular CDR in Table 4. In other specific embodiments, the CDR including the binding site is 5 to 10, 5 to 15, 5 to 20, 11 to 15, 11 to 20, 11 to 25, or 16 to 25 amino acids in length. In other embodiments, the CDR including the binding site is at least 5, 10, 15, or 20 amino acids or is more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or 100 amino acids in length.

[0128] In specific embodiments, the modified antibody of the present invention contains less than a complete variable region, i.e., where either the heavy or the light chain contains, or both the heavy and light chains contain, less than the complete set of framework regions and three CDRs such as, for example, where the variable region contains only one or two CDRs, preferably including the intervening framework regions. [0129] In other specific embodiments, the modified antibody specifically binds to the human milk fat globule antigen, and at least one of the ADRs of the modified antibody contains an amino acid sequence selected from the following: (i) Ala-Tyr-Trp-Ile-Glu (SEQ HO NO:3); (ii) Glu-Ile-Leu-Pro-Gly-Ser-Asn-Asn-Ser-Arg-Tyr-Asn-Glu-Lys-Phe- Lys-Gly (SEQ HO NO:4); (iii) Ser-Glu-Asp-Ser-Ala-Val-Tyr-Tyr-Cys-Ser-Arg-Ser-Tyr- Asp-Phe-Ala-Trp-Phe-Ala-Tyr (SEQ HO NO:5); (iv) Lys-Ser-Ser-Gln-Ser-Leu-Leu-Tyr- Ser-Ser-Asn-Gln-Lys-Ile-Tyr-Leu-Ala (SEQ HO NO:6); (v) Trp-Ala-Ser-Thr-Arg-Glu-Ser (SEQ HO NO:7); and (vi) Gln-Gln-Tyr-Tyr-Arg-Tyr-Pro-Arg-Thr (SEQ HO NO:8). [0130] H a more specific embodiment, the ADRs of the heavy chain variable region contain one or more of the amino acid sequences (i)-(iii) above, whereas the ADRs of the light chain variable region contain one or more of the amino acid sequences (iv)-(vi) above. [0131] Hi a specific embodiment, the present invention provides a modified antibody that binds human colon carcinoma-associated antigen (Rhodes, 1999, Ann. Oncol. 10 Suppl. 4:118-21) and comprises a variable region having at least one ADR containing one of the following amino acid sequences: Thr-Ala-Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn-Asp-Val-Ala (SEQ HO NO:9); Ile-Tyr-Tyr-Ala-Ser-Asn-Arg-Tyr-Thr (SEQ HO NO: 10); Phe-Ala-Gln- Gln-Asp-Tyr-Ser-Ser-Pro-Leu-Thr (SEQ HO NO: 11); Phe-Thr-Asn-Tyr-Gly-Met-Asn (SEQ HO NO: 12); Ala-Gly-Trp-Ile-Asn-Thr-Tyr-Thr-Gly-Glu-Pro-Thr-Tyr-Ala-Asp-Asp-Phe- Lys-Gly (SEQ HO NO: 13); or Ala-Arg-Ala-Tyr-Tyr-Gly-Lys-Tyr-Phe-Asp-Tyr (SEQ HO NO: 14).

[0132] The modified antibodies of the present invention can be further altered and screened to select an antibody having higher affinity or altered specificity. Antibodies having higher affinity or altered specificity for the target antigen may be generated and selected by any method known in the art. For example, but not by way of limitation, a nucleic acid molecule encoding the modified antibody can be mutagenized, either randomly, i.e., by chemical mutagenesis, or by making particular mutations at specific positions in the nucleic acid molecule by site-directed mutagenesis, and the encoded protein then screened for higher affinity or altered specificity for the target antigen. Screening can be accomplished by testing expressed proteins individually or by screening a library of mutated proteins, e.g., by phage display techniques (see, e.g., U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698, all by Ladner et al; PCT Publication WO 92/01047 by McCafferty et al. or any other phage display technique known in the art) Accordingly, in a specific embodiment, the further modified antibody of the present invention has a higher affinity for a particular antigen than the grandparent unmodified antibody or the modified antibody that has not been further modified; or the further modified antibody binds specifically to an antigen that the grandparent unmodified antibody or modified antibody that has not been further modified does not bind specifically to.

[0133] Hi another embodiment, the modified antibody or further modified antibody of the present invention exhibits a binding constant for an antigen of at least 2xl0^"7 M. H some embodiments, the modified antibody or further modified antibody has at least 2-fold, at least 4-fold, at least 6-fold, or at least 8-fold higher specificity or affinity for an antigen than an unmodified antibody that specifically binds the same antigen. [0134] Hi one embodiment, the modified antibody of the invention can be further modified in any way known in the art, in view of this disclosure, for the modification of antibodies as long as the further modification does not eliminate binding of the further modified antibody to the particular antigen. Hi particular, the further modified antibody may have, in addition to the insertion into or replacement of ADR sequences with the amino acid sequence of a heterologous peptide, one or more amino acid substitutions, deletions, insertions, or combination thereof. Such amino acid substitutions, deletions, insertions or combination thereof may be any substitution, deletion, insertion, or combination thereof, respectively, that does not eliminate the immunospecif ic binding of the modified antibody to the target antigen. For example, such amino acid substitutions can include substitution of one or more functionally equivalent amino acid residues. For example, one or more amino acid residues can be substituted by another amino acid of a similar charge or polarity that can act as a functional equivalent, resulting essentially in a silent alteration. Substitutes for an amino acid may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. [0135] Additionally, or alternatively, one or more amino acid residues within the sequence can be substituted by a non-classical amino acid or chemical amino acid analog.

Non-classical amino acids or chemical amino acid analogs include but are not limited to the isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, Abu,

2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,

3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β- alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). (See Noren et al., 1989, Science 244

(4901): 182-188; Maglary et al, 2001, J Mol Biol 307(3):755-69; Chin et al, 2003, Science

301(56351:964-7; Kohrer et al., 2003, Chem Biol 10(11): 1095-1102.

[0136] H other specific embodiments, the present invention provides functionally active fragments of a modified antibody of the present invention. "Functionally active fragment" means that the fragment can specifically bind the target antigen, as determined by any method known in the art to determine immunospecific binding (e.g., as described in

Section 5.7 infra). Such fragments include but are not limited to: F(ab')₂ fragments, which contain the variable regions of both the heavy and the light chains, the light constant region and the CHI domain of the heavy chain, which fragments can be generated by pepsin digestion of the antibody; Fab fragments, generated by reducing the disulfide bonds of an

F(ab')₂ fragment (Figure 1; King et., 1992, Biochem. J. 281:317); and Fv fragments, i.e., fragments that contain the variable region domains of both the heavy and light chains

(Reichmann and Winter, 1988, J. Mol. Biol. 203:825; King et al, 1993, Biochem. J.

290:723).

[0137] The present invention also includes the use of single chain antibodies (SCA)

(see U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423-426; Huston et al., 1988, Proc.

Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546). SCAs are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0138] Additionally, the present invention also includes the use of heavy chain and light chain dimers and diabodies, as well as single chain Fvs (scFv), and disulfide-linked

Fvs.

[0139] Hi a preferred embodiment of the present invention, the modified antibody is derived from a human monoclonal antibody. The creation of completely human monoclonal antibodies is possible through the use of transgenic mice. Transgenic mice in which the mouse antibody gene loci have been replaced with human antibody loci provide in vivo affinity-maturation machinery for the production of human antibodies. Human antibodies may also be obtained by screening libraries of human antibodies. [0140] The modified antibodies and functionally active fragments of the present invention can be derivatized by covalent attachment of any appropriate type of molecule to the modified antibody or fragment. For example, but not by way of limitation, the modified antibody molecule or functionally active fragment can be derivatized by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by addition of a known protecting blocking group, proteolytic cleavage, or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. For example, modified antibodies or functionally active fragments of the present invention can be conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387. [0141] The present invention further provides modified antibodies or functionally active fragments thereof conjugated (covalent or non-covalent) to a heterologous polypeptide (other than any heterologous peptide that may have been inserted in an ADR) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, a modified antibody or functionally active fragment of the present invention can be used to target a heterologous polypeptide to a particular cell type either in vitro or in vivo by conjugating the modified antibody or fragment to an antibody specific for a particular cell surface receptor. Modified antibodies conjugated to a heterologous polypeptide may be used in view of this disclosure in in vitro immunoassays and purification methods according to methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al, J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

[0142] H certain embodiments, the modified antibody or functionally active fragment of the present invention is fused via a covalent bond (such as, for example, a peptide bond) at either the N-terminus or the C-terminus to an amino acid sequence of another protein or peptide that is different from the modified antibody or functionally active fragment. Hi one embodiment, the modified antibody or functionally active fragment is covalently linked to the other protein or peptide at the N-terminus of the constant domain of the modified antibody or functionally active fragment between the variable and constant domains. Hi one embodiment, the present invention provides a fusion protein in which the modified antibody or functionally active fragment is covalently linked to a portion of a growth enhancing factor or a portion of an immunostimulatory factor, such as, e.g., any of interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-10, interleukin-12, interleukin-15, G-colony stimulating factor, tumor necrosis factor, porin, interferon-gamma, NK cell antigen, and MHC derived peptide, among others. [0143] The present invention further provides compositions comprising heterologous polypeptides conjugated to fragments of modified antibodies or functionally active fragments of the present invention. For example, the heterologous polypeptide may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, or portion thereof. Methods for conjugating polypeptides to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; and 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 9 1/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337- 11341(1992) (said references incorporated by reference in their entireties). [0144] A modified antibody or functionally active fragment of the present invention can be fused to a marker sequence that facilitates purification. Many such marker sequences are commercially available. H one embodiment, the marker sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA). As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification are available, including but not limited to the hemagglutinin"HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the Flag® Epitope Tag from Affinit Bioreagents™.

[0145] The present invention further provides modified antibodies or functionally active fragments thereof conjugated to a diagnostic agent, e.g., to detect a complex that forms with another member of the binding pair in vivo. The diagnostic agent can be a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance can be conjugated either directly to the antibody (or fragment thereof) or indirectly through an intermediate (such as, for example, a linker) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. An example of a luminescent material is luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include ¹²⁵1, ¹³¹I, ^luIn and ⁹⁹Tc. [0146] The present invention further provides modified antibodies or functionally active fragments thereof conjugated to a therapeutic agent such as a cytotoxin, (e.g., a cytostatic or cytocidal agent), or a radioactive metal ion (e.g., an alpha emitter). A cytotoxin includes any agent that is detrimental to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents can also be selected from antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine); alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin); anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)); and anti-mitotic agents (e.g., vincristine and vinblastine), among others. [0147] The therapeutic agent is not to be construed as limited to classical chemical therapeutic agents. For example, the therapeutic agent may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent (e.g., TNF-α, or TNF-β), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or a biological response modifier such as, e.g., a lymphokine (e.g., interleukin-1 ("IL- 1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM- CSF"), or granulocyte colony stimulating factor ("G-CSF")), or a growth factor (e.g., growth hormone ("GH")).

[0148] Techniques for conjugating a therapeutic agent to an antibody are known in the art, see, e.g., Arnon et al., "Monoclonal Antibodies For Hnmunotargeting Of Drags Hi Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Hie. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents H Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-58.

5.2 METHODS OF PRODUCING THE MODIFIED ANTIBODY

[0149] The modified antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, in view of this disclosure.

[0150] Hi one embodiment, modified antibodies or functionally active fragments thereof of the present invention engineered in accordance with the ADR rules of the present invention are recombinantly expressed at high levels in mammalian or other eukaryotic cells, e.g. myeloma cells, CHO cells, etc., to produce at least 5 ng antibodies/L of media; at least 10 ng/mL, at least 15 ng/mL, at least 50 ng/mL, at least 100 ng/mL, at least 400 ng/L, at least 800 ng L, at least 10 μg L, at least 50 μg/L, at least 100 μg/L, at least 500 μg/L, at least 700 μg/L, at least 10 mg/L, at least 100 mg/L, at least 400 mg/L, preferably at least

700 mg/L, most preferably at least 1000 mg/L of media (and may produce as much as 2g/L,

3g/, 4g/L). The expressed modified antibody or functionally active fragment thereof of the present invention exhibits proper folding and assembly upon recombinant production as compared to the parental antibody or "parental" functionally active fragment thereof of the present invention. Hi one embodiment, modified antibodies engineered in accordance with the present invention are recombinantly expressed at a higher level relative to an unmodified antibody (e.g., a parent antibody). Antibodies engineered in accordance with the methods of the present invention can be recombinantly expressed in yields that are at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or 20-fold increased as compared to the yield of the parental antibody otherwise using the same recombinant system and expression conditions.

[0151] Recombinant expression of the modified antibody or functionally active fragment thereof of the present invention may require construction of a nucleic acid molecule encoding the modified antibody. Such a nucleic acid can be produced in view of this disclosure using general methods known in the art such as, for example, recombinant techniques or chemical synthesis (e.g., see Creighton, 1983, "Proteins: Structures and

Molecular Principles", W.H. Freeman & Co., N.Y., pp.34-49; and Sambrook et al., 1989,

Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.), or using PCR techniques on known immunoglobulin genes to engineer the appropriate nucleotide sequence.

[0152] A nucleic acid molecule that encodes a modified antibody or functionally active fragment thereof of the present invention may be assembled from chemically synthesized oligonucleotides. See, e.g., Kutmeier et al., 1994, Biotechniques 17:242, which describes the synthesis of a set of overlapping oligonucleotides containing portions of a sequence encoding a modified antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0153] Alternatively, a nucleic acid molecule containing a nucleotide sequence encoding a modified antibody, or functionally active fragment thereof of the present invention (e.g., a heavy or light chain), can be constructed from a nucleic acid molecule containing a nucleotide sequence encoding an antibody molecule or functionally active fragment thereof of the present invention. For example, nucleic acid molecules containing nucleotide sequences encoding heavy or light chains can be obtained either from existing clones of heavy or light chains or variable domains, or by isolating a nucleic acid molecule encoding a heavy or light chain or variable domain from a suitable source, e.g., an antibody

DNA library or a cDNA library prepared from cells or tissue expressing a repertoire of antibody molecules or a synthetic antibody library (see, e.g., Clackson et al., 1991, Nature

352:624; Hane et al, 1997, Proc. Natl. Acad. Sci USA 94:4937).

[0154] Accordingly, the present invention further provides nucleic acid molecules comprising a nucleotide sequence encoding a modified antibody of the invention or a heavy or light chain (or other functionally active fragment) of the modified antibody of the present invention. Hi certain embodiments, the nucleic acid molecule of the present invention is isolated.

[0155] Once a nucleic acid molecule containing a nucleotide sequence encoding at least a variable region of a heavy or light chain has been cloned, then a specific heterologous peptide-encoding sequence can be introduced into that portion of the nucleotide sequence coding for an ADR. Alternatively, a nucleic acid molecule containing a nucleotide sequence encoding a CDR sequence from a different antibody can be introduced into that portion of the nucleotide sequence coding for an ADR. Such engineering of the particular ADR coding sequence can be accomplished using general recombinant DNA techniques known in the art in view of the present disclosure. For example, the nucleotide sequence encoding the ADR can be replaced by a nucleotide sequence encoding both the ADR and the particular heterologous peptide or CDR sequence, for example, using PCR based methods, or in vitro site directed mutagenesis, etc. If a convenient restriction enzyme site is available in the nucleotide sequence encoding the ADR, then the sequence can be cleaved with the restriction enzyme and a nucleic acid molecule containing a nucleotide sequence encoding the heterologous peptide or CDR sequence can be ligated into the restriction site using standard techniques. The nucleic acid molecule encoding the heterologous peptide or CDR sequence can be obtained either from a naturally occurring nucleic acid molecule or can be generated synthetically. [0156] Hi a specific embodiment, the present invention provides a method of producing a nucleic acid encoding the modified antibody of the invention which comprises: (a) synthesizing a set of oligonucleotides, said set comprising oligonucleotides containing a portion of the nucleotide sequence that encodes the modified antibody and oligonucleotides containing a portion of the nucleotide sequence that is complementary to the nucleotide sequence that encodes the modified antibody, and each of said oligonucleotides having overlapping terminal sequences with another oligonucleotide of said set, except for those oligonucleotides containing the nucleotide sequences encoding the N-terminal and C- terminal portions of the modified antibody; (b) allowing the oligonucleotides to hybridize to each other; and (c) ligating the hybridized oligonucleotides, such that a nucleic acid containing the nucleotide sequence encoding the modified antibody is produced. [0157] Hi another embodiment, the present invention provides a method of producing a modified antibody that specifically binds a first member of a binding pair, which binding pair consists of said first member and a second member, said antibody comprising a variable region having at least one ADR containing a portion of said second member, said portion containing a binding site for said first member, said method comprises: (a) growing a recombinant cell containing a nucleic acid produced as described above such that the encoded modified antibody is expressed by the cell; and (b) recovering the expressed modified antibody. [0158] A nucleic acid molecule encoding a modified antibody or functionally active fragment thereof of the present invention optionally contains a nucleotide sequence encoding a leader sequence that directs the secretion of the modified antibody or fragment thereof of the present invention.

[0159] Once a nucleic acid molecule encoding at least the heavy or light chain variable domain of the modified antibody is obtained, it can be isolated and used to generate the heavy or light chain, or the entire antibody, e.g., by introducing the nucleic acid molecule into a vector containing a nucleotide sequence encoding the constant region of the antibody (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464). Vectors containing the complete heavy or light chain for co- expression with a nucleic acid molecule to allow the expression of a complete antibody molecule are available in the art. See, for example, pMRROlO.l and pGammal (see also Bebbington, 1991, Methods in Enzymology 2:136-145). The nucleic acid molecule can also be used to produce any desired fragment of the modified antibody. Techniques to recombinantly produce Fab, Fab', F(ab')₂ fragments can be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., 1992, BioTechniques 12:864-869; Sawai et al., 1995, AJRI 34:26-34; and Bittner et al., 1988, Science 240: 1041-1043. The nucleic acid molecule can also be used to produce human antibodies. Such recombinant production can be carried out in any desired host, including mammalian cells, insect cells, plant cells, yeast cells and bacterial cells. [0160] A nucleic acid molecule of the present invention can be transferred to a cloning or expression vector using standard recombinant techniques. An expression vector will typically contain the nucleic acid molecule in operative association with appropriate regulatory sequences that allow for the protein encoded by the nucleic acid molecule to be successfully expressed under appropriate conditions. The vector (or nucleic acid molecule) can be transferred to a host cell by conventional techniques. Cells genetically engineered to contain such vector or nucleic acid molecule can be cultured by conventional techniques so as to produce the modified antibody or functionally active fragment thereof of the present invention. Specifically, once the nucleic acid molecule encoding the modified antibody or functionally active fragment thereof has been generated, the modified antibody or fragment thereof can be expressed by methods known in the art. See also Bebbington, 1991, Methods in Enzymology 2:136-145. For example, an expression vector containing a nucleic acid molecule encoding the modified antibody or functionally active fragment thereof can be transiently transfected into COS cells, the cells cultured for an appropriate period of time to permit antibody or fragment expression, the supernatant containing the secreted antibody or fragment collected, and the antibody or fragment harvested therefrom. [0161] A variety of host-expression vector systems can be utilized to express an antibody- or fragment-coding sequence of the present invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be expressed and the antibody or fragment subsequently purified. These systems include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody or fragment coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody or fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the antibody or fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody or fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, and 3T3 NSO, Per. Cle., SP210 cells), particularly, myeloma cell lines harboring recombinant expression constracts containing antibody or fragment coding sequences.

[0162] The expression elements of vectors vary in their strengths and specificities.

Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0163] The expression of a modified antibody or functionally active fragment of the present invention may be controlled by any appropriate promoter or enhancer element known in the art. Examples of promoters which may be used to control the expression of a nucleotide sequence encoding a modified antibody or fragment of the invention include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), the tetracycline (Tet) promoter (Gossen et al, 1995, Proc. Nat. Acad. Sci. USA 89:5547-5551); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region, which is active in pancreatic beta cells (Hanahan,

1985, Nature 315:115-122); immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al, 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495); albumin gene control region, which is active in liver (Pinkert et al, 1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control region, which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al, 1987, Science 235:53-58); alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region, which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al,

1986, Cell 46:89-94); myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283- 286); neuronal-specific enolase (NSE), which is active in neuronal cells (Morelli et al , 1999, Gen. Virol. 80:571-83); brain-derived neurotrophic factor (BDNF) gene control region, which is active in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res. Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter, which is active in astrocytes (Gomes et al, 1999, Braz J Med Biol Res 32(5):619-631; Morelli et al, 1999, Gen. Virol. 80:571-83); and gonadotropic releasing hormone gene control region, which is active in the hypothalamus (Mason et al, 1986, Science 234:1372-1378).

[0164] Hi a specific embodiment, the expression of a modified antibody or fragment of the present invention is regulated by a constitutive promoter. Hi another embodiment, the expression of a modified antibody or fragment of the present invention is regulated by an inducible promoter. Hi another embodiment, the expression of a modified antibody or fragment of the present invention is regulated by a tissue-specific promoter.

[0165] Hi bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the modified antibody or fragment being expressed. For example, when a large quantity of such a protein is to be produced, e.g., for the generation of pharmaceutical compositions of the antibody or fragment, a vector directing the expression of a high level of fusion protein product that is readily purifiable may be desired. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody or fragment coding sequence can be ligated into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Hiouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109;

Van Heeke & Schuster, 1989, J. Biol Chem. 264:5503-5509); and the like. pGEX vectors may also be used to express the modified antibody or fragment of the present invention as a fusion protein with glutathione S-transferase (GST). Hi general, such fusion proteins are soluble and easily purified from lysed cells by adsorption and binding to matrix glutathione- agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0166] Hi an insect system, Autographa californica nuclear polyhedrosis virus

(AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The modified antibody or fragment coding sequence can be cloned individually into a non-essential region (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

[0167] Hi addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.

To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, Per.C6, VERO,

BHK, HeLa, COS, MDCK, 293, 3T3, WI38.

[0168] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the modified antibody or functionally active fragment thereof may be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter and enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.

Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the modified antibody or functionally active fragment.

[0169] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell ,11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci.

USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl Acad. Sci. USA 77:3567; O'Hare et al.,

1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

[0170] The expression levels of the modified antibody or functionally active fragment thereof can be increased by vector amplification (for a review, see Bebbington and

Hentschel, Tlie Use of Vectors Based on Gene Amplification for the Expression of Cloned

Genes in Mammalian Cells in DNA Cloning, Vol.3. (Academic Press, New York, 1987)).

When a marker in the vector system expressing the modified antibody or functionally active fragment thereof is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody- or fragment-encoding nucleotide sequence, production of the antibody or fragment, respectively, will also increase (Crouse et al., 1983,

Mol. Cell. Biol. 3:257).

[0171] The host cell can be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers enabling equal expression of heavy and light chain polypeptides. Alternatively, a single vector can be used encoding both heavy and light chain polypeptides. Hi such situations, the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:562; Kohler, 1980, Proc. Natl. Acad.

Sci. USA 77:2197). The coding sequences for the heavy and light chains can each comprise cDNA or genomic DNA.

[0172] The present invention further provides a recombinant host cell containing a nucleic acid molecule having a nucleotide sequence that encodes a modified antibody or functionally active fragment of the present invention. [0173] Once a modified antibody or functionally active fragment of the present invention has been produced by recombinant expression, it can be purified by any standard method known in the art for purification of a protein or immunoglobulin molecule, including, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Furthermore, the modified antibody or functionally active fragment of the present invention may be fused to a heterologous polypeptide sequence described herein or otherwise known in the art so as to facilitate purification.

5.3 ASSAYS OF THE MODIFIED ANTIBODD3S

[0174] The present invention further provides methods of characterizing a modified antibody or functionally active fragment produced according to the present invention, which methods generally involve monitoring the integrity, stability and/or purity of the expressed antibody or fragment. For example, in certain embodiments of the invention, SDS-PAGE can be used to assess purity; size exclusion high performance liquid chromatography can be used to test for integrity and aggregation; activity or biological assays can be used to determine efficacy and/or potency; ultraviolet absorbance can be used to assess concentration; and isotyping assays can be used for identification. Furthermore, enzyme- linked immunoabsorbant assay (ELISA) and/or Western-blotting can be used to determine affinity and/or avidity of a modified antibody or fragment for its cognate antigen. Finally, the present invention encompasses methods of characterizing an antibody or functionally active fragment thereof by determining its primary, secondary, or tertiary structure; its carbohydrate content; its charge isoforms; and its hydrophobic interactions. [0175] There are various methods available for assessing the stability of the modified antibody or functionally active fragment of the present invention based on the physical and chemical stractures of the antibody or fragment, as well as on its biological and immunological activity. For example, to study denaturation of the antibody or fragment, methods such as charge-transfer absorption, thermal analysis, fluorescence spectroscopy, circular dichroism, NMR, and HPSEC defined earlier, are available. See, for example, Wang et al, 1988, J. of Parenteral Science & Technology 42(supp):S4-S26. [0176] The stability of the modified antibody or functionally active fragment of the present invention can also be assessed by assays that measure the biological activity of the antibody or fragment. For example, the biological activity of an antibody can include, but is not limited to, antigen-binding activity, complement-activation activity, Fc-receptor binding activity, and so forth. Antigen-binding activity of an antibody or an antigen-binding fragment thereof can be measured by any method known in the art, including but not limited to ELIS A, radioimmunoassay, Western blot, and the like. Complement-activation activity can be measured by a C3a/C4a assay in a system where an antibody of the present invention is reacted with cells expressing its cognate antigen in the presence of the complement components. Also see Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety). An ELIS A based assay can be used to compare the ability of an antibody or functionally active fragment of the present invention to specifically bind to its cognate antigen relative to a reference standard.

[0177] The purity of the modified antibody or functionally active fragment of the present invention can be measured by any method known in the art such as, e.g., HPSEC. [0178] A modified antibody or functionally active fragment of the present invention can be assayed for its ability to specifically bind to a particular antigen. Such an assay may be performed in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991, Nature 354:82-84), on chips (Fodor, 1993, Nature 364:555-556), on bacteria (U.S. Patent No. 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al, 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310) (each of these references is incorporated herein in its entirety by reference).

[0179] Immunoassays that can be used to analyze immunospecific binding and cross- reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunoelectrophoresis assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are routine in the art (see, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).

[0180] Modified antibodies and functionally active fragments of the present invention can also be assayed using any surface plasmon resonance ("SPR") based assays known in the art for characterizing the kinetic parameters of antibody-antigen interaction.

Any commercially available SPR instrument can be used, including, but not limited to, BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsys instruments available from Affinity Sensors (Franklin, MA.); IBIS system available from Windsor Scientific Limited (Berks, UK); SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan); and SPR Detector Spreeta available from Texas Instruments (Dallas, TX). For a review of SPR-based technology see Mullet et al, 2000, Methods 22: 77-91; Dong et al, 2002, Review in Mol Biotech., 82: 303-23; Fivash et al, 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al, 2000, Current Opinion in Biotechnology 11: 54-61; all of which are incorporated herein by reference in their entirety. Additionally, any of the SPR instruments and SPR based methods for measuring protein- protein interactions described in U.S. Patent Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; and 6,268,125, all of which are incorporated herein by reference in their entireties, are contemplated for use in the methods of the invention. [0181] Hi some embodiments, where the modified antibody or functionally active fragment of the present invention binds an infectious disease agent or a cellular receptor for an infectious disease agent, and has therapeutic utility in the treatment and/or prevention of an infectious disease, the ability of the antibody or fragment to inhibit viral replication or reduce viral load can be tested in in vitro assays. A modified antibody or fragment of the present invention that binds an infectious disease agent or a cellular receptor for an infectious disease agent, and that is administered to a patient or test animal, can be assayed for its ability to inhibit or down-regulate the expression of viral polypeptides. Techniques known in the art, including but not limited to Western blot analysis, Northern blot analysis, and RT-PCR, can be used to measure the expression of viral polypeptides and/or viral titers. [0182] An antibody or fragment of the present invention can be tested in a suitable animal model system prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any appropriate animal system known in the art can be used. Temporal regime of administering the therapies (e.g., prophylactic and/or therapeutic agents) whether such therapies are administered separately or as an admixture, and the frequency of administration of the therapies may be adjusted depending on the response of the treatment. [0183] After constructing an antibody or fragment having one or more ADRs containing a binding site for a particular molecule, any appropriate binding assay known in the art can be used to assess the binding between the resulting modified antibody or fragment and the particular molecule. These assays may also be performed to select modified antibodies or fragments thereof that exhibit a higher affinity or specificity for the particular antigen than the corresponding umodified antibody or unmodified fragment. [0184] H one embodiment, the degree of binding is determined by detecting a label on the primary antibody or fragment. Hi another embodiment, the degree of binding of the antibody or fragment is detected by detecting binding of a secondary antibody or reagent to the (primary) antibody or fragment. Hi a particular embodiment, the secondary antibody is labeled. Various means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0185] An example of an in vitro assay system useful in assessing the binding of a modified antibody or functionally active fragment of the present invention to its target molecule is described below. Briefly, a reaction mixture of the modified antibody (or fragment) and a test sample containing the target molecule is incubated under conditions and for a time sufficient to allow the modified antibody (or fragment) and the target molecule in the test sample to interact, i.e., to bind to each other, thus forming a complex, which can then be detected. This assay can be conducted in a variety of ways. For example, one of the two components, i.e., the modified antibody (or fragment), or the test sample is anchored onto a solid phase medium, the solid phase medium is then contacted with the non-anchored component, and the presence of a complex of antibody (or fragment) and target molecule on the solid phase medium is then detected at the end of the reaction.

Hi one embodiment of such a method, the modified antibody (or fragment) is labeled, either directly or indirectly, and the target molecule is anchored onto a solid phase medium. Hi a specific embodiment, microtiter plates are utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished, e.g., by coating the solid phase with a solution of the target molecule and allowing the solution to air dry.

[0186] H order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed with anchored component will remain immobilized on the solid surface.

The detection of complexes remaining on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates the presence of complexes. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes present on the surface.

[0187] Alternatively, a complexing reaction can be conducted in a liquid phase without an anchoring solid phase medium, the complexes separated from unreacted components, and the complexes detected and quantified by standard techniques. 5.4 THERAPEUTIC USE OF MODIFIED ANTIBODIES [0188] The invention also provides a method for treating a disease or disorder associated with the expression or overexpression of a particular molecule, said method comprising admmistering an appropriate modified antibody or functionally active fragment of the present invention as a therapeutic molecule (termed herein a "Therapeutic") to a subject in need of such treatment. Such a Therapeutic can be either a modified antibody of the present invention or a functionally active fragment thereof, (e.g., as described in Sections 5.1 and 5.2, supra), or a nucleic acid molecule having a nucleotide sequence encoding such a modified antibody or fragment (e.g., as described in Sections 5.1 and 5.2, supra.).

[0189] H a particular embodiment, the therapeutic method of the present invention uses a modified antibody that is derived from a human antibody. Hi another embodiment, the method of the present invention uses a modified antibody that is a chimeric or humanized antibody. Hi another embodiment, the therapeutic method of the present invention uses a functionally active fragment of a modified antibody of the present invention. Hi another embodiment, the therapeutic method of the present invention uses a functionally active fragment of a modified antibody of the present invention that is a chimeric or humanized antibody.

[0190] The present invention further provides pharmaceutical compositions comprising a modified antibody or functionally active fragment of the present invention combined with a pharmaceutically active carrier. The modified antibody or fragment specifically binds a particular molecule, e.g., an antigen, thereby treating a disease or disorder associated with the expression or overexpression of the particular antigen. Hi embodiments discussed in more detail in the subsections that follow, modified antibodies (or fragments thereof) that specifically bind to a tumor or cancer antigen, or to an antigen of an infectious disease agent, or to a cellular receptor for an infectious disease agent, can be used to treat or prevent tumors, cancers, or infectious diseases associated with the expression or overexpression of the particular antigen or receptor. Modified antibodies (or fragments) that specifically bind to a ligand or receptor may be used to treat or prevent a disease associated with an abnormal increase in the amount or activity of the particular ligand receptor. Hi certain embodiments, the modified antibodies (or fragments) are used to treat or prevent an autoimmune disease, including but not limited to rheumatoid arthritis, lupus, ulcerative colitis, or psoriasis. The modified antibodies (or fragments) of the present invention may also be used to treat allergies. [0191] The subjects or patients to which the present therapeutic methods can be applied include any vertebrate species, and more specifically any mammalian species, including, but not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, monkeys, rabbits, chimpanzees, and humans. Hi a preferred embodiment, the subject is a human or companion animal.

5.4.1 TREATMENT ANDPREVENTION OF CANCERS

[0192] The present invention further provides a therapeutic method for treating a cancer characterized by the expression or over-expression of a particular cancer antigen, which antigen is a member of a binding pair. The therapeutic method includes administering to a subject in need of such treatment an appropriately selected Therapeutic that specifically binds to the particular cancer antigen and can thereby be used to treat a cancer.

[0193] Cancers include any disease or disorder characterized by uncontrolled cell growth including, but not limited to, neoplasms, tumors and metastases. Such cancers can be treated by administration of the appropriate modified antibody or fragment of the present invention, which modified antibody or fragment specifically binds to an antigen associated with cancer cells present in the cancer to be treated. Whether a particular Therapeutic is effective to treat a certain type of cancer can be determined by any method known in the art such as those methods described in Section 5.6, infra, in view of the present disclosure [0194] For example, but not by way of limitation, cancers and tumors associated with the following cancer and tumor antigens may be treated by administering the appropriate modified antibody or fragment of the present invention containing in its ADR an amino acid sequence that recognizes a cancer antigen, such as: KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415); ovarian carcinoma antigen (CA125) (Yu et al., 1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailor et al, 1990, Nucl Acids Res. 18(16):4928); prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli et al, 1993, Cancer Res. 53:227-230); carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc. Clin. Oncol. 13:294); polymorphic epithelial mucin antigen; human milk fat globule antigen; a colorectal tumor-associated antigen, such as CEA, TAG-72 (Yokata et al, 1992, Cancer Res. 52:3402-3408), CO17-1A (Ragnhammar et al, 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol 2:135), CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19 (Ghetie et al, 1994, Blood 83:1329-1336); human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445); CD33 (Sgouros et al., 1993, J. Nucl Med. 34:422-430); melanoma-associated antigen p97 (Estin et al,

1989, J. Natl. Cancer Instit. 81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al,

1990, J. Exp. Med. 171(4):1375-1380); high molecular weight melanoma antigen (HMW- MAA) (Natali et al., 1987, Cancer 59:55-63; Mittelman et al, 1990, J. Clin. Invest. 86:2136-2144); melanoma specific antigens such as ganglioside GD2 (Saleh et al., 1993, J.Hnmunol., 151, 3390-3398), ganglioside GD3 (Shitara et al, 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250); tumor- specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188); differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923); antigens of fibrosarcoma, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immuno. 141:1398-1403); neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (Epidermal growth factor receptor), HER2 antigen (pl85^HER2), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359); malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:301-304); differentiation antigen (Feizi, 1985, Nature 314:53-57) such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes and preimplantation embryos, I(Ma) found in gastric adenocarcinomas, Ml 8, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D_t56-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^y found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells , Eι series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (blood group Le^a) found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Le^b), G49 found in EGF receptor of A431 cells, MH2 (blood group ALe^b/Le^y) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, TsA found in myeloid cells, R₂ found in melanoma, 4.2, G_D3, Dl.l, OFA-1, GM₂, OFA-2, G_D2, and Ml:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos. Hi one embodiment, the antigen is a T-cell receptor-derived peptide from a cutaneous T-cell lymphoma (see, Edelson, 1998, The Cancer Journal 4:62). [0195] The Therapeutic used to treat cancer can be administered in conjunction with one or more chemotherapeutic agents including, but not limited to, methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorabicin, idarabicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etc. In one embodiment, the modified antibody or fragment is conjugated to a chemotherapeutic agent or other type of toxin, e.g., a ricin toxin, or a radionuclide, or another agent effective to kill cancer cells or to arrest cancer cell growth. H another embodiment, the modified antibody or fragment has a first ADR containing a binding site for a cancer antigen and a second ADR containing a binding site for a molecule on the surface of an immune cell, such as, but not limited to, a T-cell, a B-cell, NK-cell, K-cell, TIL-cell or neutrophil. [0196] Hi one embodiment of the present invention, the ADR of an antibody or fragment has been modified to include an amino acid sequence that specifically binds a human colon carcinoma-associated protein antigen. Hi one embodiment, the antibody or fragment has at least one of the following characteristics: (i) it recognizes an epitope of a protein component of the antigen, but does not recognize epitopes of the carbohydrate component(s) of the antigen; (ii) the antigen recognized by the modified antibody or fragment is not detectable on normal human tissue; and (iii) the antigen is detectable only on colon carcinoma cells.

[0197] Hi another embodiment of the present invention, the ADR of an antibody or fragment has been modified to include an amino acid sequence that specifically binds an antigen that is detectable only on breast carcinoma cells.

[0198] Hi yet another embodiment of the present invention, the ADR of an antibody or fragment has been modified to include an amino acid sequence that specifically binds an antigen that is detectable only on ovarian carcinoma cells. [0199] Cancers and related disorders that can be treated or prevented by administration of an appropriately selected Therapeutic of the present invention include but are not limited to those listed in Table 5 (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):

TABLE S MALIGNANCIES AND RELATED DISORDERS

Leukemia acute leukemia acute lymphocytic leukemia acute myelocytic leukemia myeloblastic promyleocytic myelomonocytic monocytic erythroleukemia chronic leukemia chronic myelocytic (granulocytic) leukemia chronic lymphocytic leukemia Polycythemia vera Lymphoma Hodgkin's disease non-Hodgkin's disease Multiple myeloma Waldenstrδm's macroglobulinemia Heavy chain disease Solid tumors sarcomas and carcinomas fibrosarcoma myxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordoma angiosarcoma endotheliosarcoma lymphangiosarcoma lymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumor leiomyosarcoma rhabdomyosarcoma colon carcinoma pancreatic cancer breast cancer ovarian cancer prostate cancer squamous cell carcinoma basal cell carcinoma adenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillary carcinoma papillary adenocarcinomas cystadenocarcinoma medullary carcinoma bronchogenic carcinoma renal cell carcinoma hepatoma __^ bile duct carcinoma choriocarcinoma seminoma embryonal carcinoma Wilms' tumor cervical cancer uterine cancer testicular tumor lung carcinoma small cell lung carcinoma bladder carcinoma epithelial carcinoma glioma astrocytoma medulloblastoma craniopharyngioma ependymoma pinealoma hemangioblastoma acoustic neuroma oligodendroglioma meningiomamelanoma neuroblastoma retinoblastoma

[0200] Hi specific embodiments, an appropriately selected Therapeutic of the present invention can be used to treat or prevent cancer or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, in the ovary, bladder, breast, colon, lung, skin, pancreas, prostate, uterus, gastrointestinal tract, B lymphocytes or T lymphocytes. Hi other specific embodiments, a sarcoma, melanoma, or leukemia is treated or prevented.

[0201] Hi other specific embodiments, an appropriately selected Therapeutic of the present invention can be used to treat a pre-malignant condition or to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 5. Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to cancer or neoplasia, particularly where non-neoplastic cell growth consisting of hyperplasia, metaplasia or dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.)

5.4.2 TREATMENT OF INFECTIOUS DISEASE

[0202] The present invention further provides a method of treating or preventing an infectious disease comprising administering to a subject in need of said treatment an appropriate Therapeutic of the present invention that specifically binds to an antigen of the infectious disease-causing agent, or a cellular receptor to which the infectious disease- causing agent binds, or an enzyme expressed by the infectious diseases agent, wherein such immunospecific binding is effective in the treatment or prevention of the infectious disease. As discussed below, infectious agents include, but are not limited to, viruses, bacteria, fungi, protozoa, and parasites.

[0203] Hi a specific embodiment, an infectious disease is treated by administration of a modified antibody or fragment thereof that specifically recognizes one of the following antigens of an infectious disease agent: influenza viras hemagglutinin (Genbank accession no. JO2132; Air, 1981, Proc. Natl. Acad. Sci. USA 78:7639-7643; Newton et al., 1983, Virology 128:495-501); human respiratory syncytial virus G glycoprotein (Genbank accession no. Z33429; Garcia et al., 1994, J. Virol; Collins et al, 1984, Proc. Natl. Acad. Sci. USA 81:7683); core protein, matrix protein or other protein of Dengue viras (Genbank accession no. M19197; Hahn et al., 1988, Virology 162:167-180); measles virus hemagglutinin (Genbank accession no. M81899; Rota et al., 1992, Virology 188:135-142); herpes simplex virus type 2 glycoprotein gB (Genbank accession no. M14923; Bzik et al., 1986, Virology 155:322-333); poliovirus I VP1 (Emini et al., 1983, Nature 304:699); an envelope glycoprotein of HJV I (Putney et al., 1986, Science 234:1392-1395); hepatitis B surface antigen (Itoh et al., 1986, Nature 308:19; Neurath et al., 1986, Vaccine 4:34); diptheria toxin (Audibert et al., 1981, Nature 289:543); streptococcus 24M epitope (Beachey, 1985, Adv. Exp. Med. Biol. 185:193); gonococcal pilin (Rothbard and Schoolnik, 1985, Adv. Exp. Med. Biol. 185:247); pseudorabies viras g50 (gpD); pseudorabies virus II (gpB); pseudorabies virus gill (gpC); pseudorabies virus glycoprotein H; pseudorabies virus glycoprotein E; transmissible gastroenteritis glycoprotein 195; transmissible gastroenteritis matrix protein; swine rotavirus glycoprotein 38; swine parvoviras capsid protein; Serpulina hydodysenteriae protective antigen; bovine viral diarrhea glycoprotein 55; Newcastle disease virus hemagglutinin-neuraminidase; swine flu hemagglutinin; swine flu neuraminidase; foot and mouth disease viras; hog colera viras; swine influenza virus; African swine fever virus; Mycoplasma hyopneumoniae; infectious bovine rhinotracheitis virus (e.g., infectious bovine rhinotracheitis viras glycoprotein E or glycoprotein G), or infectious laryngotracheitis virus (e.g., infectious laryngotracheitis virus glycoprotein G or glycoprotein I); a glycoprotein of La Crosse virus (Gonzales-Scarano et al., 1982, Virology 120:42); neonatal calf diarrhea virus (Matsuno and Hiouye, 1983, Infection and Immunity 39:155); Venezuelan equine encephalomyelitis viras (Mathews and Roehrig, 1982, J.

Immunol. 129:2763); punta toro virus (Dalrymple et al., 1981, in Replication of Negative Strand Viruses, Bishop and Compans (eds.), Elsevier, NY, p. 167); murine leukemia virus (Steeves et al., 1974, /. Virol. 14:187); mouse mammary tumor viras (Massey and Schochetman, 1981, Virology 1_.15:20); hepatitis B virus core protein and/or hepatitis B viras surface antigen or a fragment or derivative thereof (see, e.g., U.K. Patent Publication No. GB 2034323A published June 4, 1980; Ganem and Varmus, 1987, Ann. Rev. Biochem. 56:651-693; Tiollais et al, 1985, Nature 317:489-495); antigen of equine influenza viras or equine herpesvirus (e.g., equine influenza viras type A/Alaska 91 neuraminidase, equine influenza viras type A/Miami 63 neuraminidase; equine influenza virus type A/Kentucky 81 neuraminidase; equine herpesvirus type 1 glycoprotein B; equine herpesvirus type 1 glycoprotein D); antigen of bovine respiratory syncytial virus or bovine parainfluenza virus (e.g., bovine respiratory syncytial viras attachment protein (BRSV G); bovine respiratory syncytial virus fusion protein (BRSV F); bovine respiratory syncytial viras nucleocapsid protein (BRSV N); bovine parainfluenza virus type 3 fusion protein; and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase); bovine viral diarrhea virus glycoprotein 48 or glycoprotein 53.

[0204] Cellular receptors that can be targeted for treatment of an infectious disease are listed in Table 6 below, along with the pathogen that binds to that cellular receptor.

TABLE 6

[0205] Viral diseases that can be treated using a Therapeutic of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenoviras, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinoviras, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova viras, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV- II), any picornaviridae, entero viruses, caliciviridae, any of the Norwalk group of viruses, togaviruses, such as Dengue virus, alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebola viruses, parainfluenza virus, orthomyxo viruses, bunyavirases, arenaviruses, reovirases, rotaviruses, orbiviruses, human T cell leukemia viras type I, human T cell leukemia virus type II, simian immunodeficiency virus, lentiviruses, polyomavirases, parvoviruses, Epstein-Barr viras, human herpesviras-6, cercopithecine herpes viras 1 (B virus), poxvirases, and encephalitis.

[0206] Bacterial diseases that can be treated using a Therapeutic of the present invention include those caused by, but not limited to, gram negative or gram positive bacteria, mycobacteria rickettsia, mycoplasma, Shigella spp., Neisseria spp. (e.g., Neisseria mennigitidis and Neisseria gonorrhoeae), legionella, Vibrio cholerae, Streptococci, such as Streptococcus pneumoniae, corynebacteria diphtheriae, clostridium tetani, bordetella pertussis, Haemophilus spp. (e.g., influenzae), Chlamydia spp., Enterotoxigenic Escherichia coli, etc. and bacterial diseases Syphillis, Lyme's disease.

[0207] Protozoal diseases that can be treated using a Therapeutic of the present invention include those caused by, but not limited to, plasmodia, Eimeria, Leishmannia, kokzidioa, and trypanosoma, and fungi such as Candida.

[0208] Hi one embodiment, the Therapeutic of the present invention is administered in combination with an appropriate antibiotic, antifungal, anti-viral or other drag useful in treating the particular disease. Hi one embodiment, the Therapeutic is conjugated to a compound effective against the infectious disease-causing agent to which the Therapeutic is directed such as, for example, an antibiotic, anti-fungal or anti- viral agent. Hi another embodiment, the Therapeutic has a first ADR containing a binding site for an antigen of an infectious disease agent, and a second ADR containing a binding site for a molecule on the surface of an immune cell, such as but not limited to a T-cell, a B-cell, NK-cell, TIL-cell or neutrophil.

[0209] Hi an alternative embodiment, a nucleic acid molecule comprising a nucleotide sequence encoding a Therapeutic of the present invention is administered to a subject to treat a disease or disorder associated with the expression of a molecule to which the Therapeutic specifically binds.

5.5 PHARMACEUTICAL PREPARATIONS AND METHODS OFADMINISTRATION

[0210] A Therapeutic of the present invention can be formulated for administration to a subject in any conventional manner using one or more physiologically acceptable carriers or excipients.

[0211] Various routes may be used to administer the Therapeutic of the present invention including, but not limited to pulmonary, sublingual, intrathecal, parenteral, mucosal, rectal, vaginal, oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes.

[0212] Thus, a modified antibody (or functionally active fragments thereof, or corresponding nucleic acid molecule) can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or by intravenous, intramuscular, nasal, sublingual, intrathecal, mucosal, vaginal, intracerebral, intradermal, intraperitoneal, oral, buccal, parenteral or rectal administration.

[0213] For oral administration, the Therapeutic can take the form of, for example, a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), and/or wetting agents (e.g., sodium lauryl sulphate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, e.g., solutions, syrups or suspensions, or they can be presented as a dry product for re-constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils), and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents, as appropriate.

[0214] Preparations for oral administration can be suitably formulated to give immediate release or controlled release of the Therapeutic as appropriate.

[0215] For buccal administration, the Therapeutic can take the form of a tablet or lozenge formulated in a conventional manner.

[0216] For administration by inhalation, the Therapeutic can be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Hi the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0217] For parenteral administration, the Therapeutic can be formulated for intravenous or intramuscular injection, via, for example, bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, optionally with an added preservative. The composition can take the form of a suspension, solution or emulsion in oily or aqueous vehicles, and can contain one or more formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for re-constitution with a suitable vehicle, e.g., sterile pyrogen-free water.

[0218] For rectal administration, the Therapeutic can be formulated as a suppository, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0219] The Therapeutic can be formulated as a depot preparation. Such long acting formulation can be administered by implantation (for example, subcutaneously or intramuscularly), or by intramuscular injection. Thus, e.g., the Therapeutic can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative.

[0220] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[0221] The precise dose of the Therapeutic to be employed in the formulation will depend on the route of administration, and the nature and needs of the particular patient, and should be decided according to the judgment of the practitioner and each patient's circumstances according to standard clinical techniques. An effective amount of the

Therapeutic is that amount sufficient to produce a healthful benefit in the treated subject, such as an improvement in one or more symptoms of a disease or disorder, or a slowing in the progression of the disease or disorder, after a single dose or after a course of treatment.

Where the Therapeutic is used to induce an immune response, e.g., against cancer cells, an effective amount of the Therapeutic is an amount sufficient to induce an immune response in the subject to which the vaccine preparation has been administered. Effective doses may be extrapolated from dose-response curves derived from animal model test systems.

[0222] Toxicity and therapeutic efficacy of a Therapeutic can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.

Compounds that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0223] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in a therapeutic method of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. 5.6 DIAGNOSTIC METHODS [0224] Modified antibodies and fragment of the present invention that bind to a specific antigen can be used as diagnostics. Hi various embodiments, the present invention provides the detection and quantification of a member of a binding pair, and the uses of such measurements in clinical applications. Modified antibodies or functionally active fragments thereof can be used, e.g., in the detection of an antigen in a biological sample to test a subject for aberrant levels of the antigen to which the modified antibody or fragment binds, and/or for the presence of abnormal forms of such molecules. By "aberrant levels" is meant a substantially increased or decreased level relative to a standard level in an analogous biological sample from a subject not having the particular disease or disorder. Modified antibodies and fragments of the present invention can also be included as a reagent in a kit for use in a diagnostic or prognostic technique.

[0225] A modified antibody or fragment of the present invention that specifically binds to a cancer antigen, or an antigen of an infectious disease agent, can be used to diagnose, prognose or screen for a cancer or infectious disease, respectively, associated with the expression of the particular antigen. Hi one aspect, the present invention provides a method of diagnosing or screening for the presence in a subject of a cancer characterized by the expression (or increased expression) of a particular cancer antigen. The cancer antigen can be a first member of a binding pair. The method can comprise measuring the level of specific binding of a modified antibody or fragment of the present invention to a cancer antigen present in a biological sample derived from the subject, in which the modified antibody or fragment specifically binds to the cancer antigen.

[0226] Hi another embodiment, the present invention provides a method for detecting abnormal levels of a target molecule (i.e., a particular ligand or receptor) in a biological sample derived from a subject comprising comparing the level of specific binding of a modified antibody or fragment of the present invention, which antibody or fragment specifically binds to the target molecule, to the level of immunospecific binding of the modified antibody or fragment of the present invention to the target molecule in a standard or control sample.

[0227] The measurement of a molecule that is bound by a modified antibody or fragment of the present invention can be used to detect and/or stage a disease or disorder related to the expression or overexpression of the molecule in a subject, in screening of such disease in a population of subjects, in differential diagnosis of the physiological condition of the subject, and in monitoring the effect of a therapeutic treatment on the subject. [0228] The following assays are presented as non-limiting examples of methods to detect molecules to which the modified antibodies or fragments of the present invention specifically bind.

[0229] Hi a specific embodiment, a diagnostic method is used to detect abnormalities in the level of gene product, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular expression of the particular molecule to be assayed. [0230] The binding activity of a given modified antibody or fragment can be determined according to methods known in the art in view of the present disclosure. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

[0231] One of the ways in which a modified antibody or fragment can be detectably labeled is by linking the antibody or fragment to an enzyme for use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., 1978, J. Clin. Pathol 31:507-520; Butler, 1981, Meth. Enzymol 73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo)). The enzyme bound to the modified antibody will typically react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or other means. Enzymes that can be used to detectably label the modified antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholrnesterase. The detection can be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

[0232] Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the modified antibody or fragment thereof, it is possible to detect the protein that the antibody was designed for through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, 1986, Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

[0233] It is also possible to label the modified antibody or fragment with a fluorescent compound. When the fluorescently labeled antibody or fragment is exposed to light of the proper wave-length, its presence can be detected through fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0234] The modified antibody or fragment can also be detectably labeled using fluorescence-emitting metals such as Eu, or others of the lanthanide series. These metals can be attached to the antibody or fragment using such metal chelating groups as diethylenetriaminepentacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).

[0235] The modified antibody or fragment can alternatively be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent- tagged antibody or fragment can then be determined by detecting the presence of luminescence that arises during the course of an appropriate chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0236] Likewise, a bioluminescent compound can be used to label the modified antibody or fragment of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Examples of bioluminescent compounds used for purposes of labeling are luciferin, luciferase and aequorin.

[0237] The present invention further provides in vivo detection, i.e., methods of using a modified antibody or fragment of the present invention to detect the location or concentration of binding pairs. Such detection can be carried out by any method available in the art. For example, but not by way of limitation, the first member of a binding pair can be detected by administering a modified antibody or fragment (i.e., the second member of the binding pair) containing a binding domain specific for the first member of the binding pair within an ADR, and which antibody or fragment is further conjugated to a label that is detectable by the particular imaging method to be used. Such an in vivo imaging methods can be used to image a tumor or other cancerous tissue in vivo where the modified antibody or fragment that specifically binds to an antigen specific for that tumor, is further detectably labeled. 5.7 DEMONSTRATION OF THERAPEUTIC UTILITY [0238] Where the Therapeutic is a modified antibody or functionally active fragment of the specific invention that recognizes a cancer antigen, the potential efficacy of the modified antibody or fragment can be assayed by contacting the Therapeutic with cells (either from a patient or from a cultured cancer cell line), and assaying for cell survival or growth using any method known in the art. For example, cell proliferation can be assayed by measuring ³H-thymidine incorporation, or by direct cell count, or by detecting changes in transcriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers. Trypan blue staining or other standard technique can be used to assess cell viability. Differentiation can be assessed visually based on changes in morphology, etc. [0239] Where the Therapeutic is a modified antibody or functionally active fragment of the present invention that recognizes an antigen specific to an infectious disease-causing agent, or specific to a cellular receptor for an infectious disease-causing agent, the potential therapeutic efficacy of the modified antibody or fragment can be assessed by: (i) contacting the Therapeutic with cells (either from a subject or from a cultured cell line) that are infected with the infectious disease-causing agent; and (ii) assaying the cells for a reduction in the load of infectious disease-causing agent or for a reduction in any physiological indicator of infection with that particular infectious disease-causing agent. [0240] Alternatively, the the potential therapeutic efficacy of the Therapeutic can be assessed by: (i) contacting the Therapeutic with cells (either from a subject or from a cultured cell line) that are susceptible to infection by the infectious disease-causing agent, but that are not currently infected with the infectious disease-causing agent; (ii) exposing the cells to the infectious disease-causing agent under conditions that would permit infection of the cells with the infectious disease-causing agent; and (iii) determining whether the infection rate of the cells contacted with the Therapeutic is lower than the infection rate of cells not so contacted with the Therapeutic. The rate of cells infected with an infectious disease agent can be determined by any method known in the art. [0241] Where the Therapeutic is a modified antibody or functionally active fragment of the present invention that specifically binds to one of the two members of a ligand:receptor pair, the potential therapeutic efficacy of the modified antibody or fragment can be tested by: (i) contacting the Therapeutic with cells (either from a patient or from a cultured cell line) that express the member of the ligand:receptor pair to which the antibody or fragment specifically binds; and (ii) determining whether the Therapeutic prevents ligand binding to the receptor or receptor signaling, or if the Therapeutic stimulates receptor signaling. These indicators can be measured by any method known in the art for measuring ligandreceptor binding and/or receptor activity.

[0242] The Therapeutic can also be tested for potential therapeutic efficacy in an appropriate animal model, as well as in clinical trials in humans. The efficacy of the Therapeutic can be determined by any method in the art. For example, after administration of the Therapeutic to the animal model or to the human subject, the animal or human subject is evaluated for any indicator of the disease or disorder that the Therapeutic is intended to treat. For example, the efficacy of the Therapeutic can be assessed in the animal model or human subject at a suitable time interval after the initiation of therapy by determining the level of the molecule against which the modified antibody or fragment is directed. Any change or absence of change in the amount of the molecule can be identified and correlated with the efficacy of the treatment in the subject. The level of the molecule can be determined by any method known in the art such as, e.g., by any of the immunoassay methods described in Section 5.3, supra.

[0243] Hi another aspect, the modified antibody or fragment of the present invention can be tested for efficacy by monitoring a treated subject for improvement or recovery from the particular disease or condition, or one or more symptoms associated with the disease or condition, against which the modified antibody is directed. When the modified antibody or fragment is directed against a cancer antigen, the progression of the particular cancer can be followed by any diagnostic or screening method known for monitoring a cancer. For example, but not by way of limitation, the progression of the cancer can be monitored by assaying the level of a particular cancer antigen in an appropriate biologcial sample derived from the subject.

[0244] Where the Therapeutic is specific for an antigen of an infectious disease- causing agent, or is specific for a cellular receptor of an infectious disease-causing agent, the efficacy of the Therapeutic can be assessed by: (i) administering the Therapeutic to a subject (either a human subject or an animal model of the disease); and (ii) monitoring either (a) the level of the particular infectious disease-causing agent in the subject or animal model or in a biological sample from the subject or animal model, or (b) a symptom of the particular infectious disease in the subject or animal model. The levels of the infectious disease-causing agent can be determined by any method known in the art such as, e.g., by measuring viral titer in the case of a virus, or bacterial levels (for example, by culturing a sample derived from the patient), etc. The levels of the infectious disease-causing agent can also be determined by measuring the levels of the antigen against which the Therapeutic is directed. A statistically significant decrease in the levels of the infectious disease-causing agent or an amelioration of the symptoms of the infectious disease can help demonstrate that the Therapeutic is effective.

[0245] Hi specific embodiments, the modified antibody of the present invention may be used to modulate the activity of a member of a binding pair. Hi certain embodiments, the modulation is an increased in activity of the member. Hi other embodiments, the modulation is a decreased in activity of the member.

5.8 TRANSGENIC ANIMALS [0246] The present invention also provides animals that are transgenic for (i.e., contain a nucleic acid molecule encoding) a modified antibody or functionally active fragment of the present invention. Animals of any species, including but not limited to mice, rats, rabbits, guinea pigs, sheep, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees, can be used to generate transgenic animals of the present invention.

[0247] Accordingly, in a specific embodiment, the present invention provides a non- human animal wherein one or more cells of said animal contain a recombinant nucleic acid molecule that contains a nucleotide sequence encoding a modified antibody or fragment of the present invention. For example, the present invention provides a non-human animal: (i) that is transgenic for a nucleic acid molecule having a nucleotide sequence encoding a modified antibody or fragment that specifically binds to a cancer antigen; or (ii) that is transgenic for a nucleic acid molecule having a nucleotide sequence encoding a modified antibody or fragment that specifically binds to an antigen of an infectious disease-causing agent, or to a cellular receptor of an infectious disease-causing agent. [0248] Any technique known in the art can be used to introduce the modified antibody (or fragment) transgene into an animal to produce a founder line of transgenic animals. Such techniques include but are not limited to pronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al, 1989, Cell 57:717-723); etc. For a review of such Techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol 115:171-229, which is incorporated by reference herein in its entirety.

[0249] A nucleic acid molecule encoding a modified antibody or fragment of the present invention may be present in all cells of the animal, or only in some cells of the animal (i.e., a mosaic animal". The transgene can be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene can also be selectively introduced into, and activated in, a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236) in view of this disclosure. Regulatory sequences required for such a cell- type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. If it is desired that a non- integrated nucleic acid molecule encoding an antibody transgene become integrated into the chromosomal site encoding an endogenous antibody, then gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing nucleotide sequences homologous to the endogenous antibody are designed for the purpose of integrating, via homologous recombination, with chromosomal sequences, and disrupting the function of the nucleotide sequence of the endogenous immunoglobulin gene. The transgene may also be selectively introduced into a particular cell type by following, for example, the teaching of Gu et al. (Gu et al., 1994, Science 265:103-106). Regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

[0250] Methods for the production of single-copy transgenic animals with chosen sites of integration are also known in the art (see, e.g., Bronson et al., 1996, Proc. Natl. Acad. Sci. USA 93:9067-9072).

6. EXAMPLE

6.1 MODIFIED ANTIBODIES CONTAINING SOMATOSTATIN CONSTRUCTS

[0251] Hi order to determine if modifying antibodies according to the ADR rules served to improve expression levels (as compared to monoclonal antibodies modified by standard CDR definitions), antibodies were modified to express a region of somatostatin or a peptide phage display derived mimetic of somatostatin capable of binding to the somatostatin receptor.

[0252] H the variable region of the K-light chain CDRl, the ADR rales identified five positions exhibiting framework contacts (shown in FIG. 2 as underlined amino acid residues). As shown in FIG. 2, constracts D and G contain modifications based on the ADR rales of the present invention, whereas the remaining constructs, A, B, C, E, and F contain modifications that delete one or more of the amino acid residues identified by the ADR rales as critical for variable region folding. For example, in construct D, the first six amino acids at positions 24, 25, 26, 27, 28 and 29 (i.e., R-A-S-Q-S-I (SEQ HO NO:22 (position 1-

6)) of CDRl of the kappa light chain were maintained, thereby preserving the amino acid residues identified as critical for antibody structure and function by the presently disclosed

ADR rales. A peptide phage display derived mimetic of somatostatin (i.e., C-R-F-W-K-T-

W-C (SEQ HO NO:l)) capable of binding to the somatostatin receptor was inserted in between amino acid residues at positions 29 and 30 of CDRl of the kappa light chain according to the ADR rules. Hi constract G, a portion of the somatostatin peptide (i.e. , K-N-

F-F-W-K-T-F-T-S (SEQ HO NO:2)) was inserted between the amino acid residues at positions 29 and 33, and the amino acid residues at positions 30, 31 and 32 of CDRl of the kappa light chain were modified; the residues that are critical (based on the presently disclosed ADR rales) were maintained. Constracts E and F contain modifications that follow the Chothia and Kabat CDRl definitions.

[0253] Hi constructs A, B, and C, the peptide phage display derived mimetic of somatostatin (i.e., C-R-F-W-K-T-W-C (SEQ HO NO:l)) was inserted between residues 23 and 32, 26 and 35, and 26 and 32, respectively. In constracts E and F, a portion of the somatostatin peptide (i.e., K-N-F-F-W-K-T-F-T-S (SEQ HO NO:2)) was inserted between the amino acid residues at positions 23 and 35, and 23 and 34, respectively, of CDRl of the kappa light chain. Each of these five constracts contain one or more alterations of the amino acid sequences at positions within CDRl that are considered critical for antibody structure and function according to the presently disclosed ADR rales.

[0254] Hi addition to making modifications to CDRl of the kappa light chain, two modified constracts were also prepared in CDR2 of the kappa light chain where the ADR rules identified one position exhibiting framework contacts (shown in FIG. 2 as an underlined amino acid residue). As shown in FIG. 2, a portion of the somatostatin peptide

(i.e., K-N-F-F-W-K-T-F-T-S (SEQ HO NO:2)) was inserted in constract H between the amino acid residues at positions 49 and 54 of CDR2 of the kappa light chain maintaining the residue at position 54 identified as critical for antibody structure and function by the presently disclosed ADR rales. Hi construct I, a portion of the somatostatin peptide (i.e., K-

N-F-F-W-K-T-F-T-S (SEQ HO NO:2)) was inserted in CDR2 of the kappa light chain following the Kabat CDR definitions for CDR2 of the kappa light chain.

[0255] One additional modified constract was also prepared in CDRl of the heavy chain where the ADR rules identified six positions exhibiting framework contacts (shown in

FIG. 4 as underlined amino acid residues). As shown in FIG. 4, a portion of the somatostatin peptide (i.e., K-N-F-F-W-K-T-F-T-S (SEQ HO NO:2)) was inserted in CDRl of the heavy chain between residues 29 and 34 (constract J) thereby preserving the amino acid residues identified as critical for antibody structure and function by the presently disclosed ADR rules.

6.2 EXPRESSION OF MQDDTED ANTIBODD3S

[0256] Expression plasmids containing the polynucleotides that encode the modified consensus antibodies were transiently transfected into suspension Chinese Hamster Ovary Kl (CHO-K1) cells. Duplicate co-transfections of the single gene constructs (individual heavy and light chain expression plasmids) were performed at a ratio of 3:1 (light chai heavy chain) using the cationic liposomal reagent CLONfectin™ (Clontech). Transfections were harvested at day 7, and the cell culture supernatent was filtered and assayed by ELIS A for human antibody levels using anti-human IgG sera and anti-human kappa-HRP conjugated antibody (Southern Biotechnology). Assembled antibody concentrations were multiplied by the volumes of recovered cell culture supernatent for each well and normalized to the expression level of the unmodified consensus antibody that was performed as a standard in each experiment.

[0257] FIGS. 6, 7 and 8 show the relative expression levels of assembled antibodies

(heavy chain and light chain) secreted into the media. Modified antibody expression was normalized to the expression level of the consensus antibody. Although several of the modified antibodies (constructs D, G and I) were expressed at higher levels relative to the consensus antibody in FIGS. 6 and 7 when compared to FIG. 8, the overall pattern of expression is similar in all three figures (i.e., constructs built following the ADR rales in general are expressed at higher levels than constructs that delete critical amino acid residues).

[0258] For constructs prepared in CDRl of the kappa light chain, significantly higher levels of expression were detected for constructs D and G that follow the ADR rales than for constracts A, B, C, E and F (see FIGS. 6 and 8). Antibodies in which CDRl was modified according to the Chothia and Kabat CDR definitions (constructs E and F) were expressed at levels 10 to 40-fold lower than construct G and the control consensus antibody (FIGS. 6 and 8). The only difference between constracts E and F, and construct G, is the CDRl sequence. The inability to detect normal levels of assembled antibody expressed from constracts E and F suggests a disruption in folding or stability, presumably a consequence of modifying those amino acid residues that are essential for folding or stability of the antibody. Since these residues were not modified in construct G and since expression levels from constract G were comparable to the control, adherence to the ADR rules appears to improve the expression of modified antibodies. [0259] The expression levels of modified antibodies A, B and C are also significantly lower than construct D (where the ADR rules were followed) and the consensus antibody. Hi construct A, amino acid residues 24, 25, 26 and 29 identified by the ADR rules as critical for variable region folding have been replaced with amino acids from the inserted peptide mimetic of somatostatin while residue 33 is maintained. Expression levels of the resulting modified antibody are approximately 50-fold lower than construct D (FIG. 8). Hi construct B, amino acid residues 24, 25 and 26 are maintained whereas residues 29 and 33 are replaced by amino acids from the inserted peptide mimetic of somatostatin. Hi this case, expression levels of the modified antibody are reduced approximately 30-fold when compared to constract D (FIG. 8). Interestingly, in construct C, amino acid residues 24, 25, 26 and 33 are maintained and only residue 29 is replaced. Expression levels of construct C are only 3-fold lower than constract D (FIG. 8) suggesting that replacing multiple amino acid residues identified as critical for antibody structure and function by the presently disclosed ADR rales (as in constracts A and B) can have an additive negative effect on the expression level of a modified antibody. These results corroborate the importance of amino acid residue 29 of the kappa light chain in the proper folding of the variable domain and confirm the significance of adhering to the ADR rules to improve the expression of modified antibodies.

[0260] Two modified antibodies were also prepared in CDR2 of the kappa light chain, constracts Hand I. Hi constract H, the somatostatin peptide (i.e., K-N-F-F-W-K-T-F- T-S (SEQ ID NO:2)) was inserted between amino acid residues 49 and 54 maintaining the amino acid at position 54 identified as critical for variable domain folding whereas in constract I, the somatostatin peptide was inserted between residues 49 and 57 replacing the amino acid residue at position 54. Based on the ADR rales, it was anticipated that the expression level of constract I would be lower than that of constract H. Surprisingly, the expression levels for both of these modified antibodies were found to be similar and relatively high (equivalent to the consensus antibody in FIG. 7 or approximately one half of the consensus antibody in FIG. 8). One potential explanation for these results may be that the phenylalanine of the inserted sequence (K-N-F-F-W-K-T-F-T-S (SEQ ID NO:2)) replaces the function of leucine at position 54, maintaining the presence of a hydrophobic residue in the core. This model suggests that amino acid residues in some inserted sequences may be able to mimic or compensate for amino acid residues identified as critical for antibody structure and function by the ADR rales when these critical amino acids are removed. [0261] Hi FIG. 7, the expression level is shown for a modified antibody in which the somatostatin peptide was inserted into CDRl of the heavy chain (FIG. 4, constract J). Hi construct J, the somatostatin peptide was inserted between amino acid residues 29 and 34 of CDRl of the heavy chain maintaining the six positions identified by the ADR rales that exhibit framework contacts. As expected, the expression levels of constract J are equivalent to the consensus antibody (FIG. 7) demonstrating in another CDR the importance of adhering to the ADR rales to improve the expression of modified antibodies.

6.3 CHARACTERIZATION OF BINDING OF ANTIBODIES CONTAINING SOMATOSTATIN

[0262] The ability of antibodies modified to contain somatostatin sequences to bind to membranes isolated from a cell line containing a cloned somatostatin receptor 5 (SSTR5) was tested. Hi this competition assay, varying concentrations of the natural ligand (SST- 14), the control (consensus) antibody or one of four modified antibodies (constructs D, G, I and J) were incubated with fixed amounts of membrane and radioligand (¹²⁵I-[Tyr¹ ^-SST- 14). After incubation, filtration and washing, the amount of bound radioligand was measured and recorded as decays per minute (DPM). High DPM values indicate little competition for the receptor, whereas low values are obtained when the added compound competes effectively with the radioligand for binding.

[0263] FIGS. 9 and 10 are graphic displays of the results from binding reactions performed on consecutive days. Hi each case, the data show that the natural ligand generates a sigmoidal binding curve with an EC50 value higher than the K_d determined by the membrane supplier. However, the consensus antibody, that has no somatostatin sequence inserted, exhibits no specific binding. Each of the modified antibodies tested binds in a range between these two extremes. Specifically, construct G shows a slight affinity for the membranes, but is unable to compete all the radioligand at its highest concentration. Constructs D and J compete better with the radioligand for binding to the receptor and approach the lower plateau seen in the SST-14 curve. Construct I binds to the membranes with the highest affinity of the four modified antibodies tested, suggesting that the somatostatin peptide is displayed in a conformation that allows for improved binding to SSTR5.

[0264] The above data demonstrate that modifying antibodies according to the presently disclosed ADR rules serves to improve the success of these modifications by identifying positions in the CDRs that should not be altered. The validity of this concept has been shown here with four somatostatin constructs. Each constract was able to specifically compete with a radioligand for receptor binding. When the antibodies are modified according to the ADR rules of the present invention, expression levels of the modified antibodies are improved. Importantly, the data show specific binding by modified antibodies in which the somatostatin sequence was inserted in three different CDRs (CDRl and CDR2 in the kappa light chain and CDRl in the heavy chain). Even more surprising is the increased CDR size that was tolerated in the modified antibodies. Inserts of 7 and 8 amino acids in CDRl of the kappa light chain had no deleterious effects on the expression levels of the modified antibodies. CDR2 of the kappa light chain, which rarely varies in size, accommodated three additional amino acids and heavy chain CDRl accomodated as many as six additional amino acid residues without adversely effecting the expression levels of the modified antibodies.

[0265] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0266] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A modified antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, which modified antibody specifically binds a first member of a binding pair, which binding pair consists of a first member and a second member, said modified antibody comprising a heterologous peptide contained entirely within at least one antibody display region (ADR) of said variable region, said heterologous peptide containing a portion of said second member, which portion binds said first member, and wherein said second member is not an immunoglobulin.

2. The modified antibody of claim 1 wherein said ADR which comprises said heterologous peptide is in the first CDR of the heavy chain variable region.

3. The modified antibody of claim 2 wherein said ADR consists of amino acid position 30 to amino acid position 33.

4. The modified antibody of claim 2 wherein said ADR consists of amino acid position 28 to amino acid position 33 and the amino acid residue at position 29 is hydrophobic.

5. The modified antibody of claim 2 wherein said ADR consists of amino acid position 30 to amino acid position 35 and the amino acid residue at position 34 is hydrophobic.

6. The modified antibody of claim 2 wherein said ADR consists of amino acid position 28 to amino acid position 35 and the amino acid residues at positions 29 and 34 are hydrophobic.

7. The modified antibody of claim 1 wherein said ADR which comprises said heterologous peptide is in the second CDR of the heavy chain variable region.

8. The modified antibody of claim 7 wherein said ADR consists of amino acid position 52 to amino acid position 58.

9. The modified antibody of claim 7 wherein said ADR consists of amino acid position 52 to amino acid position 62 and the amino acid residue at position 59 is tyrosine.

10. The modified antibody of claim 7 wherein said ADR consists of amino acid position 52 to amino acid position 65 and the amino acid residue at position 59 is tyrosine and the amino acid residue at position 63 is hydrophobic.

11. The modified antibody of claim 7 to 10 wherein the amino acid residue at position 51 is hydrophobic.

12. The modified antibody of claim 1 wherein said ADR which comprises said heterologous peptide is in the third CDR of the heavy chain variable region.

13. The modified antibody of claim 12 wherein said ADR consists of amino acid position 95 to amino acid position 100 or amino acid position 95 to an amino acid position that is one amino acid residue before amino acid position 101.

14. The modified antibody of claim 13 wherein the amino acid residue at amino acid position 94 and the amino acid residue at amino acid position 101 form a salt bridge.

15. The modified antibody of claim 13 wherein the amino acid residue at amino acid position 100K and the amino acid residue at amino acid position 102 together form a hydrophobic interaction.

16. The modified antibody of claim 13 wherein the heterologous peptide is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length and the amino acid residue before amino acid position 101 is hydrophobic.

17. The modified antibody of claim 1 which has a kappa light chain and wherein said ADR which comprises said heterologous peptide is in the first CDR of the kappa light chain variable region.

18. The modified antibody of claim 17 wherein said ADR consists of amino acid position 30 to amino acid position 32.

19. The modified antibody of claim 17 wherein said ADR consists of amino acid position 27 to amino acid position 32 and the amino acid residue that is two positions after amino acid position 27 is hydrophobic.

20. The modified antibody of claim 17 wherein said ADR consists of amino acid position 27 to amino acid position 32 and the amino acid residue at amino acid position 29 is hydrophobic.

21. The modified antibody of claim 17 wherein the amino acid residue at amino acid position 26 is serine.

22. The modified antibody of claim 17 wherein the amino acid residue at amino acid position 33 is hydrophobic.

23. The modified antibody of claim 1 which has a kappa light chain and wherein said ADR which comprises said heterologous peptide is in the second CDR of the kappa light chain variable region.

24. The modified antibody of claim 23 wherein said ADR consists of amino acid position 50 to amino acid position 53.

25. The modified antibody of claim 1 which has a kappa light chain and wherein said ADR which comprises said heterologous peptide is in the third CDR of the kappa light chain variable region.

26. The modified antibody of claim 25 wherein said ADR consists of amino acid position 95A to amino acid position 96.

27. The modified antibody of claim 25 wherein said ADR consists of amino acid position 92 to amino acid position 96 and the amino acid residue at amino acid position 95 is proline.

28. The modified antibody of claim 25 wherein the amino acid residue at amino acid position 89 or amino acid position 90 or both is glutamine.

29. The modified antibody of claim 25 wherein the amino acid residue at amino acid position 97 is threonine.

30. The modified antibody of claim 1 which has a lambda light chain and wherein said ADR which comprises said heterologous peptide is in the first CDR of the lambda light chain variable region.

31. The modified antibody of claim 30 wherein said ADR consists of amino acid position 25 to amino acid position 27A and amino acid position 29 to amino acid position

32.

32. The modified antibody of claim 30 wherein the amino acid residue at amino acid position 27B comprises a polar side-chain.

33. The modified antibody of claim 30 wherein said ADR consists of amino acid position 25 to amino acid position 32 and the amino acid residue at amino acid position 28 is hydrophobic.

34. The modified antibody of claim 1 which has a lambda light chain and wherein said ADR which comprises said heterologous peptide is in the second CDR of the lambda light chain variable region.

35. The modified antibody of claim 34 wherein said ADR consists of amino acid position 50 to amino acid position 53.

36. The modified antibody of claim 1 which has a lambda light chain and wherein said ADR which comprises said heterologous peptide is in the third CDR of the lambda light chain variable region.

37. The modified antibody of claim 36 wherein said ADR consists of amino acid position 91 to amino acid position 96.

38. The modified antibody of claim 36 or 37 wherein the amino acid residue at amino acid position 91 is an aromatic residue.

39. The modified antibody of claim 1, in which said portion of said second member is at least 4 amino acids in length.

40. The modified antibody of claim 1, in which said portion of said second member is in the range of 10-20 21-30, 31-40, 41-50, 51-60 or 61-70 amino acids in length.

41. The modified antibody of claim 1, in which more than one ADR contains a portion of said second member, wherein each said portion binds said first member.

42. The modified antibody of claim 1 in which the first member is a cancer antigen.

43. The modified antibody of claim 1 in which said first member is an antigen of an infectious disease agent.

44. The modified antibody of claim 1 in which said binding pair is a ligand-receptor binding pair.

45. The modified antibody of claim 1 wherein said variable region further comprises a conservative amino acid substitution at an amino acid position that is in a CDR and not in an ADR.

46. The modified antibody of claim 1 wherein said variable region has no amino acid modification at an amino acid position in a CDR that is not in an ADR.

47. An isolated nucleic acid comprising a nucleotide sequence encoding a variable region of the modified antibody of claim 1, wherein said variable region contains said ADR containing said heterologous peptide.

48. The isolated nucleic acid of claim 47 in which said nucleic acid is in a vector.

49. A recombinant cell containing the nucleic acid of claim 47.

50. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the modified antibody of claim 1; and a pharmaceutically acceptable carrier.

51. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the nucleic acid of claim 47 or the recombinant cell of claim 49; and a pharmaceutically acceptable carrier.

52. A method of improving a humanized or engineered antibody having a human or humanized light chain variable region and a human or humanized heavy chain variable region, and specifically binding an antigen, said method comprising making at least one amino acid substitution in at least one CDR of said humanized or engineered antibody, wherein said at least one amino acid substitution replaces an amino acid residue that does not conform to the ADR rules with an amino acid residue that conforms to the ADR rales, wherein said improved humanized or modified antibody specifically binds said antigen.

53. A method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region and specifically binding an antigen, said method comprising: (a) identifying at least one amino acid residue in a CDR of said humanized or engineered antibody that does not conform to the ADR rales; and (b) making an amino acid substitution at said at least one amino acid residue that does not conform to the ADR rules with an amino acid residue that conforms to the ADR rales, wherein said improved humanized or modified antibody specifically binds said antigen.

54. A method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, and specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said humanized or engineered antibody comprising a heterologous peptide contained entirely within an ADR, said heterologous peptide containing a portion of said second member, which portion binds said first member, said method comprising making at least one amino acid substitution in at least one CDR of said humanized or engineered antibody to replace an amino acid residue that does not conform to the ADR rules with an amino acid that conforms to the ADR rales, wherein said improved humanized or modified antibody specifically binds said first member.

55. A method of improving a humanized or engineered antibody having a human or humanized heavy chain variable region and a human or humanized light chain variable region, and specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said humanized or engineered antibody comprising a heterologous peptide contained entirely within an ADR, said heterologous peptide containing a portion of said second member, which portion binds said first member, said method comprising: (a) identifying at least one amino acid residue that does not conform to the ADR rules in a CDR or said humanized or engineered antibody; and (b) making an amino acid substitution at said at least one amino acid residue that does not conform to the ADR rales with an amino acid that conforms to the ADR rales, wherein said improved humanized or modified antibody specifically binds said first member.

56. The method of claim 52, 53, 54, or 55 wherein said at least one amino acid substitution is in the first CDR of the heavy chain variable region.

57. The method of claim 52, 53, 54, or 55 wherein said at least one amino acid substitution is in the second CDR of the heavy chain variable region.

58. The method of claim 52, 53, 54, or 55 wherein said at least one amino acid substitution is in the third CDR of the heavy chain variable region.

59. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a kappa light chain variable region and said at least one amino acid substitution is in the first CDR of the kappa light chain variable region.

60. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a kappa light chain variable region and said at least one amino acid substitution is in the second CDR of the kappa light chain variable region.

61. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a kappa light chain variable region and said at least one amino acid substitution is in the third CDR of the kappa light chain variable region.

62. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a lambda light chain variable region and said at least one amino acid substitution is in the first CDR of the lambda light chain variable region.

63. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a lambda light chain variable region and said at least one amino acid substitution is in the second CDR of the lambda light chain variable region.

64. The method of claim 52, 53, 54, or 55 wherein said humanized or engineered antibody has a lambda light chain variable region and said at least one amino acid substitution is in the third CDR of the lambda light chain variable region.

65. The method of claim 52, 53, 54, or 55 wherein said improved humanized or engineered antibody has increased recombinant production as compared to said humanized or engineered antibody.

66. The method of claim 52, 53, 54, or 55 wherein said improved humanized or engineered antibody has increased binding affinity, binding specificity or both, as compared to said humanized or engineered antibody.

67. A method of engineering an antibody to specifically bind a first member of a binding pair, which binding pair consists of a first member and a second member, said antibody contains a human or humanized heavy chain and a human or humanized light chain variable region, said method comprising introducing entirely within an ADR of said antibody, a heterologous peptide comprising a portion of said second member, which portion binds said first member.

68. A method of engineering an antibody to specifically binding a first member of a binding pair, which binding pair consists of a first member and a second member, said antibody contains a human or humanized heavy chain and a human or humanized light chain variable region, said method comprising: (a) identifying an ADR in said antibody; and (b) introducing entirely within said ADR, a heterologous peptide comprising a portion of said second member, which portion binds said first member.

69. The method of claim 67 or 68 wherein said ADR is in the first CDR of the heavy chain variable region.

70. The method of claim 69 wherein said ADR consists of amino acid position 30 to amino acid position 33.

71. The method of claim 69 wherein said ADR consists of amino acid position 28 to amino acid position 33 and the amino acid residue at position 29 is hydrophobic.

72. The method of claim 69 wherein said ADR consists of amino acid position 30 to amino acid position 35 and the amino acid residue at position 34 is hydrophobic.

73. The method of claim 69 wherein said ADR consists of amino acid position 28 to amino acid position 35 and the amino acid residues at positions 29 and 34 are hydrophobic.

74. The method of claim 67 or 68 wherein said ADR is in the second CDR of the heavy chain variable region.

75. The method of claim 74 wherein said ADR consists of amino acid position 52 to amino acid position 58.

76. The method of claim 74 wherein said ADR consists of amino acid position 52 to amino acid position 62 and the amino acid residue at position 59 is tyrosine.

77. The method of claim 74 wherein said ADR consists of amino acid position 52 to amino acid position 65 and the amino acid residue at position 59 is tyrosine and the amino acid residue at position 63 is hydrophobic.

78. The method of claim 74 to 77 wherein the amino acid residue at position 51 is hydrophobic.

79. The method of claim 67 or 68 wherein said ADR is in the third CDR of the heavy chain variable region.

80. The method of claim 79 wherein said ADR consists of amino acid position 95 to amino acid position 100 or amino acid position 95 to an amino acid position that is one amino acid residue before amino acid position 101.

81. The modified antibody of claim 80 wherein the amino acid residue at amino acid position 94 and the amino acid residue at amino acid position 101 form a salt bridge.

82. The modified antibody of claim 80 wherein the amino acid residue at amino acid position 100K and the amino acid residue at amino acid position 102 together form a hydrophobic interaction.

83. The modified antibody of claim 80 wherein said heterologous peptide is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length and the amino acid residue before amino acid position 101 is hydrophobic.

84. The method of claim 67 or 68 wherein said antibody has a kappa light chain variable region and said ADR is in the first CDR of the kappa light chain variable region.

85. The method of claim 84 wherein said ADR consists of amino acid position 30 to amino acid position 32.

86. The method of claim 84 wherein said ADR consists of amino acid position 27 to amino acid position 32 and the amino acid residue that is two positions after amino acid position 27 is hydrophobic.

87. The method of claim 84 wherein said ADR consists of amino acid position 27 to amino acid position 32 and the amino acid residue at amino acid position 29 is hydrophobic.

88. The method of claim 84 wherein the amino acid residue at amino acid position 26 is serine.

89. The method of claim 84 wherein the amino acid residue at amino acid position 33 is hydrophobic.

90. The method of claim 67 or 68 wherein said antibody has a kappa light chain variable region and said ADR which comprises said heterologous peptide is in the second CDR of the kappa light chain variable region.

91. The method of claim 90 wherein said ADR consists of amino acid position 50 to amino acid position 53.

92. The method of claim 67 or 68 wherein said antibody has a kappa light chain variable region and said ADR which comprises said heterologous peptide is in the third CDR of the kappa light chain variable region.

93. The method of claim 92 wherein said ADR consists of amino acid position 95A to amino acid position 96.

94. The method of claim 92 wherein said ADR consists of amino acid position 92 to amino acid position 96 and the amino acid residue at amino acid position 95 is proline.

95. The method of claim 92 wherein the amino acid residue at amino acid position 89 or amino acid position 90 or both is glutamine.

96. The method of claim 92 wherein the amino acid residue at amino acid position 97 is threonine.

97. The method of claim 67 or 68 wherein said antibody has a lambda light chain variable region and said ADR which comprises said heterologous peptide is in the first CDR of the lambda light chain variable region.

98. The method of claim 97 wherein said ADR consists of amino acid position 25 to amino acid position 27A and amino acid position 29 to amino acid position 32.

99. The method of claim 97 wherein the amino acid residue at amino acid position 27B comprises a polar side-chain.

100. The method of claim 97 wherein said ADR consists of amino acid position 25 to amino acid position 32 and the amino acid residue at amino acid position 28 is hydrophobic.

101. The method of claim 67 or 68 wherein said antibody has a lambda light chain variable region and said ADR which comprises said heterologous peptide is in the second CDR of the lambda light chain variable region.

102. The method of claim 101 wherein said ADR consists of amino acid position 50 to amino acid position 53.

103. The method of claim 67 or 68 wherein said antibody has a lambda light chain variable region and said ADR which comprises said heterologous peptide is in the third CDR of the lambda light chain variable region.

104. The method of claim 103 wherein said ADR consists of amino acid position 91 to amino acid position 96.

105. The method of claim 103 or 104 wherein the amino acid residue at amino acid position 91 is an aromatic residue.

106. The method of claim 67 or 68, in which said portion of said second member is at least 4 amino acids in length.

107. The method of claim 67 or 68, in which said portion of said second member is in the range of 10-2021-30, 31-40, 41-50, 51-60 or 61-70 amino acids in length.

108. The method of claim 67 or 68, in which more than one ADR contains a portion of said second member, wherein each said portion binds said first member.

109. The method of claim 67 or 68 in which said first member is a cancer antigen.

110. The method of claim 67 or 68 in which said first member is an antigen of an infectious disease agent.

111. The method of claim 67 or 68 in which said binding pair is a ligand-receptor binding pair.

112. The method of claim 67 or 68 wherein said method further comprising making a conservative amino acid substitution at an amino acid position that is in said CDR and not in said ADR.

113. The method of claim 67 or 68 wherein said modified antibody has no amino acid modification at a position in said CDR that is not in said ADR.

114. A method for identifying, measuring or detecting a cancer antigen in a sample to be tested, said method comprising the steps of: (a) contacting the sample to be tested with the modified antibody of claim 42 that specifically binds said cancer antigen, under conditions permitting specific binding of said modified antibody to said cancer antigen; and (b) detecting binding of said modified antibody to said cancer antigen; wherein detection of binding of said modified antibody to said cancer antigen indicates the presence of said cancer antigen in said sample.

115. A method of treating or preventing, in a subject in need of such treatment or prevention, a cancer characterized by the presence of a cancer antigen, said method comprising administering to the subject a therapeutically or prophylactically effective amount of the modified antibody of claim 42 that binds said cancer antigen.

116. A method of treating or preventing, in a subject in need of such treatment or prevention, an infectious disease, said method comprising administering to the subject a therapeutically or prophylactically effective amount of the modified antibody of claim 43 that binds said antigen.

117. A modified antibody produced by the method of claim 52, 53, 54, 55, 67, or 68.