Patent Publication Number: US-2013236448-A1

Title: Concentrated polypeptide formulations with reduced viscosity

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application No. 61/231,140, filed Aug. 4, 2009, the disclosure of which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention concerns polypeptide formulations with reduced viscosity and methods of making and using the polypeptide formulations with reduced viscosity. 
     BACKGROUND OF THE INVENTION 
     Investigating polypeptide and solution behavior in highly concentrated conditions is critical to our understanding of the stability, safety, and efficacy of biological therapeutics. Recently, the effects of increasing polypeptide concentration on the stability and safety of biological therapeutics have gained significant attention from the biotechnology industry and the Food and Drug Administration (FDA). The physicochemical stability of biological therapeutics may be negatively affected simply by increasing polypeptide concentration. Chemical instability typically follows first order kinetics with regard to concentration; however, physical instability may result in complex higher order processes. It has been shown that increasing polypeptide concentration, such as IgG concentration, increases self association of these molecules resulting in increased non-ideal solution properties and significantly affects the viscosity and rheological behavior. 
     Subcutaneous administration of high concentration biological therapeutics presents a remarkable challenge to pharmaceutical scientists. For high dose regimes, the required polypeptide concentration is often greater than 100 mg/mL potentially resulting in non-ideal solution properties, decreased stability and/or decreased manufacturability and delivery. A major challenge in the development of such formulations is their high viscosity. 
     BRIEF SUMMARY OF THE INVENTION 
     Provided herein are liquid formulations comprising (a) a polypeptide in an amount of greater than about 50 mg/mL and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) in an amount of between about 0.1% to about 50% v/v of the formulation, wherein the formulation have reduced viscosity compared to the same formulation in the absence of DMSO or DMA. 
     Also provided herein are methods of making liquid formulations comprising combining (a) a polypeptide in an amount of greater than about 50 mg/mL and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) in an amount of between about 0.1% to about 50% v/v of the formulation, wherein the formulation has reduced viscosity compared to the same formulation in the absence of DMSO or DMA comprising combining the polypeptide and DMSO or DMA. 
     Provided herein are articles of manufacture comprising a container containing a liquid formulation comprising (a) a polypeptide in an amount of greater than about 50 mg/mL and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) in an amount of between about 0.1% to about 50% v/v of the formulation, wherein the formulation has reduced viscosity compared to the same formulation in the absence of DMSO or DMA. 
     In addition, provided herein are methods of using a liquid formulation comprising (a) a polypeptide in an amount of greater than about 50 mg/mL and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) in an amount of between about 0.1% to about 50% v/v of the formulation, wherein the formulation has reduced viscosity compared to the same formulation in the absence of DMSO or DMA to treat a disease or disorder comprising administering the formulation to a subject in need thereof. 
     Also provided herein are methods of delivering a liquid formulation comprising (a) a polypeptide in an amount of greater than about 50 mg/mL and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) in an amount of between about 0.1% to about 50% v/v of the formulation, wherein the formulation has reduced viscosity compared to the same formulation in the absence of DMSO or DMA to a subject in need thereof comprising administering the formulation. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is capable of forming a secondary structure, tertiary structure, and/or quaternary structure. In some embodiments, the secondary structure is a β-sheet. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is hydrophobic. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is about 100 amino acids or greater. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide has a molecular weight of greater than about 5,000 Daltons. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is a therapeutic polypeptide. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a chimeric antibody, humanized antibody, or human antibody. In some embodiments, the monoclonal antibody is an IgG monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment. In some embodiments, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv, a Fv, and a diabody. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide includes one or more of these parameters. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, DMSO or DMA is in an amount of between about 1% to about 10% v/v of the formulation. In some embodiments, DMSO or DMA is in an amount of between about 1% to about 5% v/v of the formulation. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the formulation further comprises histidine. In some embodiments, histidine is in an amount of between about 10 mM to about 100 mM. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the formulation further comprises arginine-HCl. In some embodiments, arginine-HCl is in an amount of between about 50 mM to about 200 mM. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the polypeptide is in an amount of about 100 mg/mL or greater. In some embodiments, the polypeptide is in an amount of between about 100 mg/mL and about 300 mg/mL. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, viscosity is reduced compared to the same formulation in the absence of DMSO or DMA by between about 1 to about 1000 cP. In some embodiments, the viscosity is reduced compared to the same formulation in the absence of DMSO or DMA by between about 5 to about 100 cP. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the viscosity is reduced compared to the same formulation in the absence of DMSO or DMA by between about 1.2 and about 10 fold. In some embodiments, the viscosity is reduced compared to the same formulation in the absence of DMSO or DMA by between about 1.2 and about 5 fold. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the viscosity is about 50 cP or less. In some embodiments, the viscosity is about cP or less. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the pH is between about 5 and about 8. In some embodiments, the pH is between about 5 and about 6.5. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, DMSO or DMA is DMSO. In some embodiments, DMSO or DMA is DMA. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the formulation is formulated for administration by injection. In some embodiments, the formulation is formulated for administration by subcutaneous injection. 
     In some embodiments of any of the formulations, methods, and articles of manufacture, the container is a syringe. In some embodiments, the syringe is further contained within an injection device. In some embodiments, the injection device is an autoinjector. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a viscosity of 145 mg/mL anti-IFN a solutions in the presence of varying amounts of DMSO or DMA. The buffer species and concentration was histidine chloride (25, 50, and 75 mM), pH 5.4. 
         FIG. 2  shows viscosity of 140 mg/mL anti-IFN a, 25 mM histidine chloride solutions in the presence (red circles) and absence (black squares) of 10% v/v DMSO as a function of pH. 
         FIG. 3  shows viscosity of 145 mg/mL anti-IFN a, 25 mM histidine chloride, pH 5.4 in the presence and absence of varying amounts of arginine chloride and co-solvent. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. Formulations and Methods of Making of the Formulations 
     Provided herein are liquid formulations comprising (a) a polypeptide and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA), wherein the formulation has reduced viscosity compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA. Also provided herein are methods of making the formulation of liquid formulations comprising (a) a polypeptide and (b) dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA), wherein the formulation has reduced viscosity compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA comprising combining the polypeptide and DMSO or DMA. 
     DMSO is the chemical compound with the formula (CH 3 ) 2 SO. DMSO is a colorless liquid and an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. DMA is the organic compound with the formula CH 3 C(O)N(CH 3 ) 2 . DMA is colorless, water miscible, high boiling liquid and is miscible with most other solvents, although it is poorly soluble in aliphatic hydrocarbons. In some embodiment, DMSO or DMA in the polypeptide formulation is in an amount of between about any of 0.1% to 2.5%, 0.1% to 5%, 0.1% to 7.5%, 0.1% to 10%, 1% to 2.5%, 1% to 5%, 1% to 7.5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%, 1% to 40%, or 1% to 50% of the formulation. In some embodiments, DMSO or DMA of the polypeptide formulation comprising DMSO or DMA is in an amount of about any of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, DMSO or DMA is DMSO. In some embodiments, DMSO or DMA is DMA. In some embodiments, DMSO or DMA is a combination of DMSO and DMA. 
     In some embodiments, the viscosity is shear viscosity. Shear viscosity is the viscosity coefficient when the applied stress is a shear stress (valid for non-Newtonian fluids). Shear viscosity=shear stress/shear rate. 
     In some embodiments, the shear viscosity of the polypeptide formulation comprising DMSO or DMA is reduced compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA by between about any of 1 cP to 1000 cP, 1 cP to 500 cP, 1 cP to 250 cP, 1 cP to 100 cP, 1 cP to 75 cP, 1 cP to 50 cP, 1 cP to 40 cP, 1 cP to 30 cP, 1 cP to 25 cP, 1 cP to 20 cP, 1 cP to 15 cP, 1 cP to 10 cP, 5 cP to 100 cP, 5 cP to 50 cP, 5 cP to 25 cP, or 5 cP to 15 cP. The shear viscosity of the polypeptide formulation comprising DMSO or DMA may be in some embodiments reduced compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA by greater than about any of 1 cP, 5 cP, 10 cP, 15 cP, 20 cP, 25 cP, 50 cP, 100 cP, 250 cP, 500 cP, or 1000 cP. The shear viscosity of the polypeptide formulation comprising DMSO or DMA may be in some embodiments reduced compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA by about any of 1 cP, 5 cP, 10 cP, 15 cP, 20 cP, 25 cP, 50 cP, 100 cP, 250 cP, 500 cP, or 1000 cP. 
     In some embodiments, the viscosity of the polypeptide formulation comprising DMSO or DMA is reduced compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA by between about any of 1.2 fold and 5 fold, 1.2 fold and 10 fold, 1.2 fold and fold, 2 fold to 5 fold, 2 fold to 10 fold, or 2 fold to 20 fold. The viscosity of the polypeptide formulation comprising DMSO or DMA may be in some embodiments reduced compared to the same formulation in the absence (i.e., lacking) of DMSO or DMA by greater than about any of 1.2 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, or 50 fold. In some embodiments, the viscosity is shear viscosity. 
     In some embodiments, the shear viscosity of the polypeptide formulation comprising DMSO or DMA is about any of 100 cP or less, 75 cP or less, 50 cP or less, 25 cP of less, 20 cP or less, 15 cP or less, or 10 cP or less. The shear viscosity of the polypeptide formulation comprising DMSO or DMA may be in some embodiments between about any of 5 cP to 30 cP, cP to 30 cP, 10 cP to 25 cP, or 15 cP to 25 cP. 
     In some embodiments, the polypeptide in the formulation is in an amount of about any of greater than 50 mg/mL, 75 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, 200 mg/mL, 250 mg/mL, or 300 mg/mL. The polypeptide in the formulation may be in an amount of about any of between about 50 mg/mL and 300 mg/mL, 50 mg/mL and 200 mg/mL, 100 mg/mL and 300 mg/mL, 100 mg/mL and 200 mg/mL, 120 mg/mL and 300 mg/mL, 140 mg/mL and 300 mg/mL, or 160 mg/mL and 300 mg/mL. In some embodiments, the polypeptide in the formulation is in an amount of about any of 50 mg/mL, 75 mg/mL, 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, 200 mg/mL, 250 mg/mL, or 300 mg/mL. 
     The polypeptide formulations in some embodiments may be prepared for storage by mixing a polypeptide having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington&#39;s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. 
     “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. 
     Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). 
     In some embodiments, the polypeptide formulation further comprises histidine. In some embodiments, histidine is present in the polypeptide formulation in an amount of about any of 10 mM to 100 mM, 25 mM to 100 mM, 50 mM to 100 mM, 10 mM to 200 mM, 25 mM. to 200 mM, 50 mM to 200 mM, or 100 mM to 200 mM. In some embodiments, the polypeptide formulation further comprises arginine-HCl. In some embodiments, the arginine-HCl is in an amount of about any of 10 mM to 100 mM, 25 mM to 100 mM, 50 mM to 100 mM, 10 mM to 200 mM, 25 mM. to 200 mM, 50 mM to 200 mM, or 100 mM to 200 mM. 
     In some embodiments, the pH of the polypeptide formulation is between about any of and 8, 5 and 7, 5 and 6.5, 5 and 6, or 5.5 and 6. 
     In some embodiments, the polypeptide in the polypeptide formulation maintains functional activity. 
     The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. 
     The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to a polypeptide, it may be desirable to include in the one formulation, an additional polypeptide (e.g., antibody). Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. 
     Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.’ 
     As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the invention described herein include “consisting” and/or “consisting essentially of” aspects and variations. 
     II. Polypeptides 
     Provided herein are polypeptides for use in any of the polypeptide formulations with reduced viscosity and methods of making polypeptide formulations with reduced viscosity that are described herein. 
     (A) Definitions for Polypeptides 
     The term “polypeptide” as used herein means a sequence of amino acids greater than 50 amino acids. In some embodiments, the polypeptide is an antibody. “Amino acids,” as used herein, includes naturally-occurring and non-naturally occurring. Amino acids include analogs, such as pegylated, lipidized, and/or toxin conjugated analogs. 
     “Purified” polypeptide (e.g., antibody) means that the polypeptide has been increased in purity, such that it exists in a form that is more pure than it exists in its natural environment and/or when initially synthesized and/or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity. 
     The term “epitope tagged” when used herein refers to a chimeric polypeptide comprising a polypeptide fused to a “tag polypeptide.” The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about and 20 amino acid residues). 
     “Active” or “activity” for the purposes herein refers to form(s) of a polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring polypeptide, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring polypeptide and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring polypeptide. 
     The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, etc. Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. 
     “Complement dependent cytotoxicity” or “CDC” refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g. polypeptide (e.g., an antibody)) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al.,  J. Immunol. Methods  202:163 (1996), may be performed. 
     A polypeptide “which binds” an antigen of interest, e.g. a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the polypeptide is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to a “non-target” polypeptide will be less than about 10% of the binding of the polypeptide to its particular target polypeptide as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). 
     With regard to the binding of a polypeptide to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. 
     The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10 −4  M, alternatively at least about 10 −5  M, alternatively at least about 10 −6  M, alternatively at least about 10 −7  M, alternatively at least about 10 −8  M, alternatively at least about 10 −9  M, alternatively at least about 10 −10  M, alternatively at least about 10 −1  M, alternatively at least about 10 −12  M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. 
     A polypeptide that “inhibits the growth of tumor cells” or a “growth inhibitory” polypeptide is one which results in measurable growth inhibition of cancer cells. In one embodiment, growth inhibition can be measured at a polypeptide concentration of about 0.1 to about 30 μg/ml or about 0.5 nM to about 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the polypeptide. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The polypeptide is growth inhibitory in vivo if administration of the polypeptide at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to about 3 months from the first administration of the polypeptide, preferably within about 5 to about 30 days. 
     A polypeptide which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is a tumor cell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the polypeptide which induces apoptosis is one which results in about 2 to about 50 fold, preferably about 5 to about 50 fold, and most preferably about 10 to about 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay. 
     A polypeptide which “induces cell death” is one which causes a viable cell to become nonviable. Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the polypeptide is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al.  Cytotechnology  17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. 
     (B) Polypeptides 
     Provided herein are polypeptides for use in any of the polypeptide formulations with reduced viscosity and methods of making polypeptide formulations with reduced viscosity. 
     In some embodiments, the polypeptide is a therapeutic polypeptide. The therapeutic polypeptide may inhibit the growth of tumor cells, induce apoptosis, and/or induce cell death. In some embodiments, the polypeptide is an antagonist. In some embodiments, the polypeptide is an agonist. In some embodiments, the polypeptide is an antibody. 
     In some embodiments, the polypeptide is greater than about any of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, or 1,000 amino acids. In some embodiments, the polypeptide has a molecular weight of greater than any of about 5,000 Daltons, 10,000 Daltons, 15,000 Daltons, 25,000 Daltons, 50,000 Daltons, 75,000 Daltons, 100,000 Dalton, 125,000 Daltons, or 150,000 Daltons. The polypeptide may have a molecular weight between about any of 50,000 Daltons to 200,000 Daltons or 100,000 Daltons to 200,000 Daltons. Alternatively, the polypeptide for use herein may have a molecular weight of about 120,000 Daltons or about 25,000 Daltons. 
     In some embodiments, the polypeptide is capable of forming a secondary structure, tertiary structure, and/or quaternary structure. In some embodiments, the polypeptide comprises a secondary structure wherein the secondary structure is an a-helix. The polypeptide may comprise less than about any of 75%, 50%, 40%, 30%, 25%, 20%, or 10% a-helix secondary structure. In some embodiments, the polypeptide comprises a secondary structure wherein the secondary structure is a B-sheet. The polypeptide may comprise greater than about any of 25%, 50%, 60%, 70%, 75%, 80%, or 90% β-sheet secondary structure. 
     In some embodiments, the polypeptide has a low mean hydrophilicity. Hydrophilicity is defined as in Hopp, T. P. and Woods, K. R.  Proc. Natl. Acad. Sci. USA  78(6), 3824-382 (1981). Hydrophilicity is a property relating to favorable thermodynamic interactions with water. The individual amino acid hydrophilicity values determined by Hopp and Woods may be used to quantify relative hydrophilicity. In general, positive hydrophilicity values are observed for charged, and polar side chains and negative values for nonpolar side chains. In some embodiments, the polypeptide has an average hydrophilicity between about any of −3 to 1, −3 to 0, −2 to 1, −2 to 0, −1 to 1, or −1 to 0. In some embodiments, the polypeptide is an antibody, wherein one or more of the CDR regions has an average hydrophilicity less than any of about 0, −1, −2, or −3. In some embodiments, at least about any of 1, 2, 3, 4, 5, or 6 CDR regions has an average hydrophilicity less than any of about 0, −1, −2, or −3. In some embodiments, the average hydrophilicity over the six CDRs of the antibody is less than any of about 0, −1, −2, or −3. 
     In some embodiments, the polypeptide has a high hydrophobicity. Hydrophobicity is defined as in Kyte, J. and Doolittle, R. F.,  J. Mol. Bio.  157, 105-132 (1982). Hydrophobicity is indicated when there is a strong solvent-solvent (water) interactions which drive molecules that do not interact strongly with the solvent out of the solvent phase. Hydrophobicity may be measured by the partitioning of individual amino acids between an aqueous and organic phase. This partition coefficient is defined as the mole fraction of molecules in the aqueous phase relative to the mole fraction in the organic phase. Generally, positive hydropathy values are observed for nonpolar side chains, and negative hydropathy for polar and charged side chains. In some embodiments, the polypeptides have a range for average hydrophobicity between about any of 2 to −1, 2 to 1, 2 to 0, or 1 to −1. In some embodiments, the polypeptide is an antibody, wherein the CDR region has a high average hydrophobicity. In some embodiments, the CDR region has an average hydrophobicity greater than about any of 0, 1 or 2. In some embodiments, at least about any of 1, 2, 3, 4, 5, or 6 CDR regions has an average hydrophobicity greater than about any of 0, 1, or 2. In some embodiments, the average hydrophobicity of the six CDRs of the antibody is greater than about any of 0, 1 or 2. 
     In some embodiments, the polypeptide has few charged amino acid side chain groups. The charge distribution and heterogeneity may be important. Charge heterogeneity, positive hydropathy values are observed for nonpolar side chains and negative hydropathy for polar and charged side chains. In some embodiments, the polypeptide has less than about any of 40%, 35%, 30%, 25%, 20%, 15%, or 10% charged amino acid side chain groups. In some embodiments, the polypeptide is an antibody, wherein the CDR region has few charged amino acid side chain groups. In some embodiments, the CDR region has less than about any of 40%, 35%, 30%, 25%, 20%, 15%, or 10% charged amino acid side chain groups. 
     In some embodiments of the formulations, the polypeptide includes one or more of these parameters. In some embodiments, the polypeptide has few charged amino acid side chains and a high hydrophobicity. In some embodiments, the polypeptide has few charged amino acid side chains and a low mean hydrophilicity. In some embodiments, the polypeptide has a high hydrophobicity and a low mean hydrophilicity. In some embodiments, the polypeptide has a high hydrophobicity, a low mean hydrophilicity, and few charged amino acid side chains. 
     Examples of polypeptides useful in the formulations and methods described herein include mammalian polypeptides, such as, e.g., growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; a-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA, e.g., Activase™, TNKase™ Retevase™); bombazine; thrombin; tumor necrosis factor-a and -β; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-a.); serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; an integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-a and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-a, -β, and -γ, colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); a viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the above-listed polypeptides. 
     (C) Antibodies 
     The polypeptide for use in any of the polypeptide formulations with reduced viscosity and methods of making polypeptide formulations with reduced viscosity in some embodiments may be an antibody. 
     Molecular targets for antibodies encompassed by the present invention include CD proteins and their ligands, such as, but not limited to: (i) CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, CD79□ (CD79a), and CD79 (CD79b); (ii) members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and v/3 integrin, including either alpha or beta subunits thereof (e.g., anti-CD11a, anti-CD 18 or anti-CD11b antibodies); (iv) growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, 7 etc; and (v) cell surface and transmembrane tumor-associated antigens (TAA), such as those described in U.S. Pat. No. 7,521,541. 
     Other exemplary antibodies encompassed by the present invention include those selected from, and without limitation, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2/neu antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratins antibody, anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa light chains antibody, anti-lambda light chains antibody, anti-melanosomes antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody and anti-Tn-antigen antibody. 
     (i) Definitions for Antibodies 
     The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeable with antibody herein. 
     Antibodies are naturally occurring immunoglobulin molecules which have varying structures, all based upon the immunoglobulin fold. For example, IgG antibodies have two ‘heavy’chains and two ‘light’ chains that are disulphide-bonded to form a functional antibody. Each heavy and light chain itself comprises a “constant” (C) and a “variable” (V) region. The V regions determine the antigen binding specificity of the antibody, whilst the C regions provide structural support and function in non-antigen-specific interactions with immune effectors. The antigen binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a particular antigen. 
     The antigen binding specificity of an antibody is determined by the structural characteristics of the V region. The variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.,  Sequences of Proteins of Immunological Interest,  5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). 
     Each V region typically comprises three complementarity determining regions (“CDRs”, each of which contains a “hypervariable loop”), and four framework regions. An antibody binding site, the minimal structural unit required to bind with substantial affinity to a particular desired antigen, will therefore typically include the three CDRs, and at least three, preferably four, framework regions interspersed there between to hold and present the CDRs in the appropriate conformation. Classical four chain antibodies have antigen binding sites which are defined by V H  and V L  domains in cooperation. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites formed by heavy chains only. Single domain engineered immunoglobulins can be prepared in which the binding sites are formed by heavy chains or light chains alone, in absence of cooperation between V H  and V L . 
     Throughout the present specification and claims, unless otherwise indicated, the numbering of the residues in the constant domains of an immunoglobulin heavy chain is that of the EU index as in Kabat et al.,  Sequences of Proteins of Immunological Interest,  5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. The residues in the V region are numbered according to Kabat numbering unless sequential or other numbering system is specifically indicated. 
     The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.,  Sequences of Proteins of Immunological Interest,  5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). 
     The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L), 50-56 (L2) and 89-97 (L3) in the V L , and around about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V H  (Kabat et al.,  Sequences of Proteins of Immunological Interest,  5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g. residues 26-32 (L), 50-52 (L2) and 91-96 (L3) in the V L , and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in the V H  (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). 
     “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. 
     “Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. 
     Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′) 2  fragment that has two antigen-binding sites and is still capable of cross-linking antigen. 
     “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V H -V L  dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. 
     The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab′) 2  antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. 
     The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. 
     Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, d, e, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. 
     “Single-chain Fv” or “scFv” antibody fragments comprise the V H  and V L  domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V H  and V L  domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Plückthun in  The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). 
     The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,  Proc. Natl. Acad. Sci. USA,  90:6444-6448 (1993). 
     The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al.,  Nature  256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al.,  Nature  352:624-628 (1991) and Marks et al.,  J. Mol. Biol.  222:581-597 (1991), for example. 
     The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al.,  Proc. Natl. Acad. Sci. USA  81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780). 
     “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. 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 hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitution(s) as noted above. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al.,  Nature  321:522-525 (1986); Riechmann et al.,  Nature  332:323-329 (1988); and Presta,  Curr. Op. Struct. Biol.  2:593-596 (1992). 
     For the purposes herein, an “intact antibody” is one comprising heavy and light variable domains as well as an Fc region. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. 
     “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. 
     A “naked antibody” is an antibody (as herein defined) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel. 
     In some embodiments, antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors. 
     “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet,  Annu. Rev. Immunol  9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al.,  PNAS  (USA) 95:652-656 (1998). 
     “Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, the cells express at least FcγRIII and carry out ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. 
     The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and Fcγ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see Daëron,  Annu. Rev. Immunol.  15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet,  Annu. Rev. Immunol  9:457-92 (1991); Capel et al.,  Immunomethods  4:25-34 (1994); and de Haas et al.,  J. Lab. Clin. Med.  126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al.,  J. Immunol.  117:587 (1976) and Kim et al.,  J. Immunol.  24:249 (1994)). 
     (ii) Polyclonal Antibodies 
     In some embodiments, the antibodies are polyclonal antibodies. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a polypeptide that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N═C═NR, where R and R 1  are different alkyl groups. 
     Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 g or 5 μg of the polypeptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund&#39;s complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund&#39;s complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. In some embodiments, the animal is boosted with the conjugate of the same antigen, but conjugated to a different polypeptide and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as polypeptide fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. 
     (iii) Monoclonal Antibodies 
     In some embodiments, the antibodies are monoclonal antibodies. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope except for possible variants that arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies. 
     For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al.,  Nature  256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). 
     In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as herein described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding,  Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)). 
     The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. 
     In some embodiments, the myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, in some embodiments, the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al.,  Monoclonal Antibody Production Techniques and Applications pp.  51-63 (Marcel Dekker, Inc., New York, 1987)). 
     Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). 
     The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al.,  Anal. Biochem.  107:220 (1980). 
     After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding,  Monoclonal Antibodies: Principles and Practice  pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. 
     The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. 
     DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, the hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as  E. coli  cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin polypeptide, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al.,  Curr. Opinion in Immunol.  5:256-262 (1993) and Plückthun,  Immunol. Revs.,  130:151-188 (1992). 
     In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al.,  Nature  348:552-554 (1990). Clackson et al.,  Nature  352:624-628 (1991) and Marks et al.,  J. Mol. Biol.  222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al.,  Bio/Technology  10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al.,  Nuc. Acids. Res.  21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. 
     The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al.,  Proc. Natl Acad. Sci. USA  81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. 
     Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen. 
     (iv) Humanized Antibodies 
     In some embodiments, the antibodies are humanized antibodies. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al.,  Nature  321:522-525 (1986); Riechmann et al.,  Nature  332:323-327 (1988); Verhoeyen et al.,  Science  239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. 
     The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al.,  J. Immunol.  151:2296 (1993); Chothia et al.,  J. Mol. Biol.  196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al.,  Proc. Natl. Acad. Sci. USA  89:4285 (1992); Presta et al.,  J. Immunol.  151:2623 (1993)). 
     It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in some embodiments of the methods, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding. 
     (v) Human Antibodies 
     In some embodiments, the antibodies are human antibodies. As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J H ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al.,  Proc. Natl. Acad. Sci. USA  90:2551 (1993); Jakobovits et al.,  Nature  362:255-258 (1993); Bruggermann et al.,  Year in Immuno.  7:33 (1993); and U.S. Pat. Nos. 5,591,669; 5,589,369; and 5,545,807. 
     Alternatively, phage display technology (McCafferty et al.,  Nature  348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat polypeptide gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S, and Chiswell, David J.,  Current Opinion in Structural Biology  3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al.,  Nature  352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al.,  J. Mol. Biol.  222:581-597 (1991), or Griffith et al.,  EMBO J.  12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332 and 5,573,905. 
     Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275). 
     (vi) Antibody Fragments 
     In some embodiments, the antibodies are antibody fragments. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,  Journal of Biochemical and Biophysical Methods  24:107-117 (1992) and Brennan et al.,  Science  229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from  E. coli  and chemically coupled to form F(ab′) 2  fragments (Carter et al.,  Bio/Technology  10:163-167 (1992)). According to another approach, F(ab′) 2  fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a “linear antibody,” e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific. 
     In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragments are antigen binding fragments. 
     (vii) Bispecific Antibodies 
     In some embodiments, the antibodies are bispecific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes. Alternatively, a bispecific antibody binding arm may be combined with an arm that binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′) 2  bispecific antibodies). 
     Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al.,  Nature  305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al.,  EMBO J.,  10:3655-3659 (1991). 
     According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. 
     In some embodiments of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al.,  Methods in Enzymology  121:210 (1986). 
     According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. In some embodiments, the interface comprises at least a part of the C H 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. 
     Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. 
     Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al.,  Science  229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′) 2  fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. 
     Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al.,  J. Immunol.  148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al.,  Proc. Natl. Acad. Sci. USA  90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the V H  and V L  domains of one fragment are forced to pair with the complementary V L  and V H  domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al.,  J. Immunol.  152:5368 (1994). 
     Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al.,  J. Immunol.  147: 60 (1991). 
     (viii) Multivalent Antibodies 
     In some embodiments, the antibodies are multivalent antibodies. A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies provided herein can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1) n -VD2-(X2) n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain. 
     (ix) Other Antibody Modifications 
     It may be desirable to modify the antibody provided herein with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al.,  J. Exp Med.  176:1191-1195 (1992) and Shopes, B. J.,  Immunol.  148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al.,  Cancer Research  53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement mediated lysis and ADCC capabilities. See Stevenson et al.,  Anti - Cancer Drug Design  3:219-230 (1989). 
     For increasing serum half the serum half life of the antibody, amino acid alterations can be made in the antibody as described in US 2006/0067930, which is hereby incorporated by reference in its entirety. 
     (D) Polypeptide Variants and Modifications 
     Amino acid sequence modification(s) of the polypeptides, including antibodies, described herein may be used in the polypeptide formulations with reduced viscosity and methods of making the polypeptide formulations with reduced viscosity. 
     (ii) Variant Polypeptides 
     “Polypeptide variant” means a polypeptide, preferably an active polypeptide, as defined herein having at least about 80% amino acid sequence identity with a full-length native sequence of the polypeptide, a polypeptide sequence lacking the signal peptide, an extracellular domain of a polypeptide, with or without the signal peptide. Such polypeptide variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a full-length native sequence polypeptide sequence, a polypeptide sequence lacking the signal peptide, an extracellular domain of a polypeptide, with or without the signal peptide. Optionally, variant polypeptides will have no more than one conservative amino acid substitution as compared to the native polypeptide sequence, alternatively no more than about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to the native polypeptide sequence. 
     The variant polypeptide may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for a desired biological activity. These variant polypeptides with truncations, deletions, and insertions may be prepared by any of a number of conventional techniques. Desired variant polypeptides may be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding a desired variant polypeptide, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the nucleic acid fragment are employed at the 5′ and 3′ primers in the PCR. Preferably, variant polypeptides share at least one biological and/or immunological activity with the native polypeptide disclosed herein. 
     Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody. 
     For example, it may be desirable to improve the binding affinity and/or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the polypeptide. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the polypeptide (e.g., antibody), such as changing the number or position of glycosylation sites. 
     Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the polypeptide with that of homologous known polypeptide molecules and minimizing the number of amino acid sequence changes made in regions of high homology. 
     A useful method for identification of certain residues or regions of the polypeptide (e.g., antibody) that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells,  Science  244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably Alanine or Polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity. 
     Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in the Table 1 below under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in the Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Original 
                 Exemplary 
                 Preferred 
               
               
                   
                 Residue 
                 Substitutions 
                 Substitutions 
               
               
                   
                   
               
             
            
               
                   
                 Ala (A) 
                 Val; Leu; Ile 
                 Val 
               
               
                   
                 Arg (R) 
                 Lys; Gln; Asn 
                 Lys 
               
               
                   
                 Asn (N) 
                 Gln; His; Asp, Lys; Arg 
                 Gln 
               
               
                   
                 Asp (D) 
                 Glu; Asn 
                 Glu 
               
               
                   
                 Cys (C) 
                 Ser; Ala 
                 Ser 
               
               
                   
                 Gln (Q) 
                 Asn; Glu 
                 Asn 
               
               
                   
                 Glu (E) 
                 Asp; Gln 
                 Asp 
               
               
                   
                 Gly (G) 
                 Ala 
                 Ala 
               
               
                   
                 His (H) 
                 Asn; Gln; Lys; Arg 
                 Arg 
               
               
                   
                 Ile (I) 
                 Leu; Val; Met; Ala; 
                 Leu 
               
               
                   
                   
                 Phe; Norleucine 
               
               
                   
                 Leu (L) 
                 Norleucine; Ile; Val; 
                 Ile 
               
               
                   
                   
                 Met; Ala; Phe 
               
               
                   
                 Lys (K) 
                 Arg; Gln; Asn 
                 Arg 
               
               
                   
                 Met (M) 
                 Leu; Phe; Ile 
                 Leu 
               
               
                   
                 Phe (F) 
                 Trp; Leu; Val; Ile; 
                 Tyr 
               
               
                   
                   
                 Ala; Tyr 
               
               
                   
                 Pro (P) 
                 Ala 
                 Ala 
               
               
                   
                 Ser (S) 
                 Thr 
                 Thr 
               
               
                   
                 Thr (T) 
                 Val; Ser 
                 Ser 
               
               
                   
                 Trp (W) 
                 Tyr; Phe 
                 Tyr 
               
               
                   
                 Tyr (Y) 
                 Trp; Phe; Thr; Ser 
                 Phe 
               
               
                   
                 Val (V) 
                 Ile; Leu; Met; Phe; 
                 Leu 
               
               
                   
                   
                 Ala; Norleucine 
               
               
                   
                   
               
            
           
         
       
     
     Substantial modifications in the biological properties of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger,  Biochemistry  second ed., pp. 73-75, Worth Publishers, New York (1975)): 
     (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M) 
     (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) 
     (3) acidic: Asp (D), Glu (E) 
     (4) basic: Lys (K), Arg (R), His(H) 
     Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: 
     (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; 
     (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 
     (3) acidic: Asp, Glu; 
     (4) basic: His, Lys, Arg; 
     (5) residues that influence chain orientation: Gly, Pro; 
     (6) aromatic: Trp, Tyr, Phe. 
     Non-conservative substitutions will entail exchanging a member of one of these classes for another class. 
     Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment). 
     A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and target. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. 
     Another type of amino acid variant of the polypeptide alters the original glycosylation pattern of the antibody. The polypeptide may comprise non-amino acid moieties. For example, the polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in the host cell or host organism, or may be a deliberate modification arising from human intervention. By altering is meant deleting one or more carbohydrate moieties found in the polypeptide, and/or adding one or more glycosylation sites that are not present in the polypeptide. 
     Glycosylation of polypeptide is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. 
     Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). 
     Removal of carbohydrate moieties present on the polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases. 
     Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. 
     (ii) Chimeric Polypeptides 
     The polypeptide described herein may be modified in a way to form chimeric molecules comprising the polypeptide fused to another, heterologous polypeptide or amino acid sequence. In some embodiments, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. 
     In an alternative embodiment, the chimeric molecule may comprise a fusion of the polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”). As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous polypeptide (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. 
     The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH 2  and CH 3 , or the hinge, CH 1 , CH 2  and CH 3  regions of an IgG1 molecule. 
     (iii) Polypeptide Conjugates 
     The polypeptide for use in polypeptide formulations with reduced viscosity and methods of making polypeptide formulations with reduced viscosity may be conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). 
     Chemotherapeutic agents useful in the generation of such conjugates can be used. In addition, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from  Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,  Aleurites fordii  proteins, dianthin proteins,  Phytolaca americana  proteins (PAPI, PAPII, and PAP-S),  momordica charantia  inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated polypeptides. Examples include  212 Bi,  131 I,  131 In,  90 Y, and  186 Re. Conjugates of the polypeptide and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al.,  Science  238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the polypeptide. 
     Conjugates of a polypeptide and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein. 
     Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub  Maytenus serrata . Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and derivatives and analogues thereof are also contemplated. There are many linking groups known in the art for making polypeptide-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020. The linking groups include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred. 
     The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hyrdoxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue. 
     Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, e.g., U.S. Pat. No. 5,712,374. Structural analogues of calicheamicin which may be used include, but are not limited to, γ 1   I , a 2   I , .a 3   I , N-acetyl-γ 1   I , PSAG and θ 1   I . Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through polypeptide (e.g., antibody) mediated internalization greatly enhances their cytotoxic effects. 
     Other antitumor agents that can be conjugated to the polypeptidesof the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex, as well as esperamicins. 
     In some embodiments, the polypeptide may be a conjugate between a polypeptide and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase). 
     In yet another embodiment, the polypeptide (e.g., antibody) may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pre-targeting wherein the polypeptide receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide). 
     In some embodiments, the polypeptide may be conjugated to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent) to an active anti-cancer drug. The enzyme component of the immunoconjugate includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form. 
     Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as  serratia  protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as “abzymes”, can be used to convert the prodrugs of the invention into free active drugs. 
     (iv) Other 
     Another type of covalent modification of the polypeptide comprises linking the polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in  Remington&#39;s Pharmaceutical Sciences,  16th edition, Oslo, A., Ed., (1980). 
     (D) Obtaining Polypeptides for Use in the Formulations and Methods 
     The polypeptides used in the formulations and methods described herein may be obtained using methods well-known in the art, including the recombination methods. The following sections provide guidance regarding these methods. 
     (i) Polynucleotides 
     “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. 
     Polynucleotides encoding polypeptides may be obtained from any source including, but not limited to, a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Accordingly, polynucleotides encoding polypeptide can be conveniently obtained from a cDNA library prepared from human tissue. The polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis). 
     For example, the polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only a heavy chain variable region (V H ) but also a heavy chain constant region (C H ), which typically will comprise three constant domains: C H 1, C H 2 and C H 3; and a “hinge” region. In some situations, the presence of a constant region is desirable. 
     Other polypeptides which may be encoded by the polynucleotide include antigen-binding antibody fragments such as single domain antibodies (“dAbs”), Fv, scFv, Fab′ and F(ab′) 2  and “minibodies”. Minibodies are (typically) bivalent antibody fragments from which the C H 1 and C K  or C L  domain has been excised. As minibodies are smaller than conventional antibodies they should achieve better tissue penetration in clinical/diagnostic use, but being bivalent they should retain higher binding affinity than monovalent antibody fragments, such as dAbs. Accordingly, unless the context dictates otherwise, the term “antibody” as used herein encompasses not only whole antibody molecules but also antigen-binding antibody fragments of the type discussed above. Preferably each framework region present in the encoded polypeptide will comprise at least one amino acid substitution relative to the corresponding human acceptor framework. Thus, for example, the framework regions may comprise, in total, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions relative to the acceptor framework regions. 
     Suitably, the polynucleotides described herein may be isolated and/or purified. In some embodiments, the polynucleotides are isolated polynucleotides. 
     The term “isolated polynucleotide” intended to indicate that the molecule is removed or separated from its normal or natural environment or has been produced in such a way that it is not present in its normal or natural environment. In some embodiments, the polynucleotides are purified polynucleotides. The term purified is intended to indicate that at least some contaminating molecules or substances have been removed. 
     Suitably, the polynucleotides are substantially purified, such that the relevant polynucleotides constitutes the dominant (i.e., most abundant) polynucleotides present in a composition. 
     Recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain may be used in the methods as described herein. By definition such nucleic acids comprise coding single stranded nucleic acids, double stranded nucleic acids consisting of said coding nucleic acids and of complementary nucleic acids thereto, or these complementary (single stranded) nucleic acids themselves. 
     Modification(s) may also be made outside the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a mutant nucleic acid may be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence may be a degenerate sequence. Degenerate sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly yeast, bacterial or mammalian cells, to obtain an optimal expression of the heavy chain variable domain and/or the light chain variable domain. 
     Sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a “homologous sequence(s)”). Here, the term “homologue” means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences. Here, the term “homology” can be equated with “identity”. 
     In some embodiments, homologous amino acid sequence and/or nucleotide sequence should encode a polypeptide which retains the functional activity and/or enhances the activity of the antibody. In some embodiments, homologous sequence is taken to include an amino acid sequence which may be at least 75, 85, or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions). In some embodiments, it is preferred to express homology in terms of sequence identity. 
     In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85, or 90% identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide described herein (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties/functions). In some embodiments, it is preferred to express homology in terms of sequence identity. 
     These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the polypeptide. 
     (ii) Expression of Polynucleotides 
     The description below relates primarily to production of polypeptides by culturing cells transformed or transfected with a vector containing polypeptide-encoding polynucleotides. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al.,  Solid - Phase Peptide Synthesis  W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,  J. Am. Chem. Soc.  85:2149-2154 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer&#39;s instructions. Various portions of the polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired polypeptide. 
     Polynucleotides as described herein are inserted into an expression vector(s) for production of the polypeptides. The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. 
     A polynucleotide is “operably linked” when it is placed into a functional relationship with another polynucleotide sequence. For example, nucleic acids for a presequence or secretory leader is operably linked to nucleic acids for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. 
     For antibodies, the light and heavy chains can be cloned in the same or different expression vectors. The nucleic acid segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. 
     Selection Gene Component— 
     Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362). In some embodiments, selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. 
     One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin. 
     Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the nucleic acid encoding antibodies described herein, such as DHFR, thymidine kinase, metallothionein-I and -III, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. 
     For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096). 
     Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding a polypeptide described herein, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199. 
     A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al.,  Nature  282:39 (1979)). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene. 
     In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of  Kluyveromyces  yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for  K. lactis . Van den Berg,  Bio/Technology  8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of  Kluyveromyces  have also been disclosed. Fleer et al.,  Bio/Technology  9:968-975 (1991). 
     Signal Sequence Component— 
     The polypeptides may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. A signal sequence can be substituted with a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, a factor leader (including  Saccharomyces  and  Kluyveromyces  a-factor leaders), or acid phosphatase leader, the  C. albicans  glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. 
     The nucleic acid sequence for such precursor region is ligated in reading frame to the nucleic acid sequence encoding the polypeptide described herein. 
     Origin of Replication— 
     Both expression and cloning vectors contain a polynucleotide sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter). 
     Promoter Component— 
     Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the polypeptide. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide. 
     Suitably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS cells—such as COS 7 cells—or CHO cells). Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the cross-reacting antibodies. 
     Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors. 
     Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 
     Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters. 
     The transcription of the polypeptides described herein from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. 
     The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,  Nature  297:598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. 
     Enhancer Element Component— 
     Transcription of a DNA encoding the anti-oxidized LDL polypeptide described herein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv,  Nature  297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5′ or 3′ to the polypeptide-encoding sequence, but is preferably located at a site 5′ from the promoter. 
     Transcription Termination Component— 
     Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein. 
     The vectors containing the polynucleotide sequences (e.g., the variable heavy and/or variable light chain encoding sequences and optional expression control sequences) can be transferred into a host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. (See generally Sambrook et al.,  Molecular Cloning: A Laboratory Manual  (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. 
     When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes,  Protein Purification  (Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity is most preferred, for pharmaceutical uses. 
     (iii) Constructs 
     Typically the construct will be an expression vector allowing expression, in a suitable host, of the polypeptide(s) encoded by the polynucleotide. The construct may comprise, for example, one or more of the following: a promoter active in the host; one or more regulatory sequences, such as enhancers; an origin of replication; and a marker, preferably a selectable marker. The host may be a eukaryotic or prokaryotic host, although eukaryotic (and especially mammalian) hosts may be preferred. The selection of suitable promoters will obviously depend to some extent on the host cell used, but may include promoters from human viruses such as HSV, SV40, RSV and the like. Numerous promoters are known to those skilled in the art. 
     The construct may comprise a polynucleotide which encodes a polypeptide comprising three light chain hypervariable loops or three heavy chain hypervariable loops. Alternatively the polynucleotide may encode a polypeptide comprising three heavy chain hypervariable loops and three light chain hypervariable loops joined by a suitably flexible linker of appropriate length. Another possibility is that a single construct may comprise a polynucleotide encoding two separate polypeptides—one comprising the light chain loops and one comprising the heavy chain loops. The separate polypeptides may be independently expressed or may form part of a single common operon. 
     The construct may comprise one or more regulatory features, such as an enhancer, an origin of replication, and one or more markers (selectable or otherwise). The construct may take the form of a plasmid, a yeast artificial chromosome, a yeast mini-chromosome, or be integrated into all or part of the genome of a virus, especially an attenuated virus or similar which is non-pathogenic for humans. 
     The construct may be conveniently formulated for safe administration to a mammalian, preferably human, subject. Typically, they will be provided in a plurality of aliquots, each aliquot containing sufficient construct for effective immunization of at least one normal adult human subject. 
     The construct may be provided in liquid or solid form, preferably as a freeze-dried powder which, typically, is rehydrated with a sterile aqueous liquid prior to use. 
     The construct may be formulated with an adjuvant or other component which has the effect of increasing the immune response of the subject (e.g., as measured by specific antibody titer) in response to administration of the construct. 
     (iv) Vectors 
     The term “vector” includes expression vectors and transformation vectors and shuttle vectors. 
     The term “expression vector” means a construct capable of in vivo or in vitro expression. 
     The term “transformation vector” means a construct capable of being transferred from one entity to another entity—which may be of the species or may be of a different species. If the construct is capable of being transferred from one species to another—such as from an  Escherichia coli  plasmid to a bacterium, such as of the genus  Bacillus , then the transformation vector is sometimes called a “shuttle vector”. It may even be a construct capable of being transferred from an  E. coli  plasmid to an  Agrobacterium  to a plant. 
     Vectors may be transformed into a suitable host cell as described below to provide for expression of a polypeptide encompassed in the present invention. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. 
     The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. Vectors may contain one or more selectable marker genes which are well known in the art. 
     These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. 
     (v) Host Cells 
     The host cell may be a bacterium, a yeast or other fungal cell, insect cell, a plant cell, or a mammalian cell, for example. 
     The invention also provides a transgenic multicellular host organism which has been genetically manipulated so as to produce a polypeptide in accordance with the invention. The organism may be, for example, a transgenic mammalian organism (e.g., a transgenic goat or mouse line). 
     Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as  E. coli . Various  E. coli  strains are publicly available, such as  E. coli  K12 strain MM294 (ATCC 31,446);  E. coli  X1776 (ATCC 31,537);  E. coli  strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as  Escherichia , e.g.,  E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella , e.g.,  Salmonella typhimurium, Serratia , e.g.,  Serratia marcescans , and  Shigella , as well as Bacilli such as  B. subtilis  and  B. licheniformis  (e.g.,  B. licheniformis  41P),  Pseudomonas  such as  P. aeruginosa , and  Streptomyces . These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant polynucleotide product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding polypeptides endogenous to the host, with examples of such hosts including  E. coli  W3110 strain 1A2, which has the complete genotype tonA;  E. coli  W3110 strain 9E4, which has the complete genotype tonA ptr3;  E. coli  W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan′;  E. coli  W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan′;  E. coli  W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an  E. coli  strain having mutant periplasmic protease. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable. 
     In these prokaryotic hosts, one can make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. 
     Eukaryotic microbes may be used for expression. Eukaryotic microbes such as filamentous fingi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors.  Saccharomyces cerevisiae  is a commonly used lower eukaryotic host microorganism. Others include  Schizosaccharomyces pombe; Kluyveromyces  hosts such as, e.g.,  K. lactis  (MW98-8C, CBS683, CBS4574),  K. fragilis  (ATCC 12,424),  K. bulgaricus  (ATCC 16,045),  K. wickeramii  (ATCC 24,178),  K. waltii  (ATCC 56,500),  K. drosophilarum  (ATCC 36,906),  K. thermotolerans , and  K. marxianus; yarrowia  (EP 402,226);  Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces  such as  Schwanniomyces occidentalis ; and filamentous fungi such as, e.g.,  Neurospora, Penicillium, Tolypocladium , and  Aspergillus  hosts such as  A. nidulans , and  A. niger . Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of  Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis , and  Rhodotorula. Saccharomyces  is a preferred yeast host, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. 
     In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce the polypeptides as described herein and in some instances are preferred (See Winnacker, From  Genes to Clones  VCH Publishers, N.Y., N.Y. (1987). For some embodiments, eukaryotic cells (e.g., COS7 cells) may be preferred, because a number of suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art, and include CHO cell lines, various Cos cell lines, HeLa cells, preferably, myeloma cell lines, or transformed B-cells or hybridomas. 
     In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/−DHFR(CHO or CHO-DP-12 line); mouse sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). 
     Alternatively, polypeptide-coding sequences can be incorporated into transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin. 
     Alternatively, the antibodies described herein can be produced in transgenic plants (e.g., tobacco, maize, soybean and alfalfa). Improved ‘plantibody’ vectors (Hendy et al.,  J. Immunol. Methods  231:137-146 (1999)) and purification strategies coupled with an increase in transformable crop species render such methods a practical and efficient means of producing recombinant immunoglobulins not only for human and animal therapy, but for industrial applications as well (e.g., catalytic antibodies). Moreover, plant produced antibodies have been shown to be safe and effective and avoid the use of animal-derived materials. Further, the differences in glycosylation patterns of plant and mammalian cell-produced antibodies have little or no effect on antigen binding or specificity. In addition, no evidence of toxicity or HAMA has been observed in patients receiving topical oral application of a plant-derived secretory dimeric IgA antibody (see Larrick et al.,  Res. Immunol.  149:603-608 (1998)). 
     Host cells are transfected or transformed with expression or cloning vectors described herein for polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in  Mammalian Cell Biotechnology: a Practical Approach  M. Butler, ed. (IRL Press, 1991). 
     Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl 2 , CaPO 4 , liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride or electroporation is generally used for prokaryotes. Infection with  Agrobacterium tumefaciens  is used for transformation of certain plant cells, as described by Shaw et al.,  Gene  23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb,  Virology  52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al.,  J. Bact.  130:946 (1977) and Hsiao et al.,  Proc. Natl. Acad. Sci . ( USA ) 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al.,  Methods in Enzymology  185:527-537 (1990) and Mansour et al.,  Nature  336:348-352 (1988). 
     Polypeptides, e.g., antibodies, can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in  E. coli  is faster and more cost efficient. For expression of polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,840,523, which describes translation initiation region (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the  E. coli  cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells. 
     Suitable host cells for the expression of glycosylated polypeptides described herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as  Spodoptera frugiperda  (caterpillar),  Aedes aegypti  (mosquito),  Aedes albopictus  (mosquito),  Drosophila melanogaster  (fruitfly), and  Bombyx mori  have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of  Autographa californica  NPV and the Bm-5 strain of  Bombyx mori  NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of  Spodoptera frugiperda  cells. 
     The host cells used to produce the polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham&#39;s F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco&#39;s Modified Eagle&#39;s Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. 
     (vi) Purification of Polypeptides 
     When using recombinant techniques, the polypeptides can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. 
     The polypeptides may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of the polypeptides can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. 
     It may be desired to purify the polypeptides from recombinant cell polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the polypeptide. Various methods of polypeptide purification may be employed and such methods are known in the art. 
     If the polypeptide is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al.,  Bio/Technology  10: 163-167 (1992) describe a procedure for isolating polypeptides which are secreted to the periplasmic space of  E. coli . Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the polypeptide is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available polypeptide concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. 
     The polypeptide composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al.,  J. Immunol. Meth.  62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al.,  EMBO J.  5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a C H 3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for polypeptide purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the polypeptide to be recovered. 
     Following any preliminary purification step(s), the mixture comprising the polypeptide of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt). 
     V. Methods of Using the Formulations 
     The formulations provided herein may be used in methods of delivering a polypeptide formulation described herein to a subject in need thereof comprising administering the formulation to a subject in need thereof. 
     Also provided herein are methods to treat, ameliorate, and/or delay progression of a disease or disorder comprising administering a formulation described herein to a subject in need thereof. 
     In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the disease or disorder is a “cell proliferative disorder” and “proliferative disorder”. In some embodiments, the disease or disorder is a tumor. In some embodiments the disease or disorder is cancer and the polypeptide is an antibody. 
     In some embodiments, the polypeptide is administered in an effective amount. In some embodiments, the polypeptide is administered in a growth inhibitory amount. In some embodiments, the polypeptide is administered in a cytotoxic amount. 
     As used herein, “treat”, “treatment,” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: decreasing one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life. 
     As used herein, “delaying” the progression means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. 
     In some embodiments, the methods of treatment described herein ameliorate (e.g., reduce incidence of, reduce duration of, reduce or lessen severity of) of one or more symptoms of the disease. 
     A “subject” herein is a mammal. In some embodiments, the mammal is a human. 
     A “symptom” is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the subject. 
     The term “effective amount” refers to an amount of a polypeptide to treat, ameliorate, and/or delay progression of a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. 
     A “growth inhibitory amount” of a polypeptide, is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “growth inhibitory amount” of a polypeptide for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. 
     A “cytotoxic amount” of a polypeptide is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “cytotoxic amount” of a polypeptide for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner. 
     The terms “cancer” and “cancerous” refer to or describe the physiological condition in subject that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases. 
     The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. 
     “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. 
     The polypeptide formulation may be administered to a subject, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, the polypeptide formulation is administered by subcutaneous injection. 
     For the treating, amelioration, and/or delaying progression of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of polypeptide will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the polypeptide formulation is administered previous therapy, the patient&#39;s clinical history and response to the polypeptide formulation, and the discretion of the attending physician. The polypeptide formulation is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of polypeptide can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to about 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. 
     Other therapeutic regimens may be combined with the administration of the polypeptide formulation. The polypeptide formulation can be used alone, or in combination therapy with, e.g., hormones, immunosuppressives, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. The polypeptide formulation can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. 
     Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent), consecutive administration in any order, and sequentially in any order. 
     The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, consecutive administration in either order, and sequential administration in any order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect. 
     VI. Articles of Manufacture 
     The polypeptide formulations described herein may be contained within an article of manufacture. The article of manufacture may comprise a container containing the polypeptide formulation. Preferably, the article of manufacture comprises: (a) a container comprising a composition comprising the polypeptide formulation described herein within the container; and (b) a package insert with instructions for administering the formulation to a subject. 
     The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a formulation and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the polypeptide. The label or package insert indicates that the composition&#39;s use in a subject with specific guidance regarding dosing amounts and intervals of polypeptide and any other drug being provided. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further contained within an injection device. In some embodiments, the injection device is an autoinjector. 
     A “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products. 
     Further details of the invention are illustrated by the following non-limiting Examples. The disclosures of all references in the specification are expressly incorporated herein by reference. 
     EXAMPLES 
     The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. 
     Example 1 
     Effects of Dimethyl Sulfoxide and Dimethylacetamide on Polypeptide Solution Viscosity 
     To investigate the effects of Dimethyl Sulfoxide and Dimethylacetamide on polypeptide solution viscosity, the following experiments were performed. 
     Materials and Methods 
     Multiple IgG1 full length monoclonal antibodies comprised of K-light chains constructed from identical human frameworks were used in this study. These antibodies were cloned, expressed in Chinese Hamster Ovary cell lines, and purified at Genentech (South San Francisco, Calif.). All reagents were ACS grade. 
     Unless otherwise stated, rhuMAb anti-IFNa was used as the starting material. All polypeptides were buffer exchanged into the appropriate conditions using Slide-a-lyzer 10,000 MWCO dialysis cassettes (Thermo Scientific Pierce) for at least 24 hours at 2-8° C. After removal from the cassette, the pH of individual solutions was measured under ambient conditions using a Mettler Toledo SevenMulti pH meter. Polypeptide concentration was determined with gravimetrically-prepared samples using a HP 8453 spectrophotometer at 280 nm and 320 nm. Density was measured using an Anton Paar DMA 5000 densitometer at 25.00° C. 
     Approximately, 500 μL sample aliquots were prepared by spiking in the co-solvent (DMSO or DMA) depending on the desired volume-volume percentage. A control without co-solvent was measured but containing equal amounts of respective formulation buffer added to maintain equal polypeptide concentration was measured for comparison. 
     Viscosity of all formulations was measured using an Anton Paar Physica MCR300 rheometer with a CP25-1 24.972 mm cone. The measurement temperature was controlled at QC using a Peltier plate. Three independent and separate 75 μl samples of each formulation were measured 20 times during 100 second time intervals, with a shear rate of 1000/second. 
     Results and Discussion 
     The shear viscosity of multiple polypeptide solutions was measured in the presence and absence of various buffer systems and polar solvents. The addition of relatively low volume to volume percents (1-10%) of DMSO and/or DMA decreases solution viscosity to varying extents ( FIG. 1 ). Interestingly, the buffer component histidine chloride reduces the viscosity of the solution at high ionic strength, however, DMSO and DMA are shown to further decrease viscosity ( FIG. 1 ). 
     The polar solvents, DMSO and DMA, decrease the solution viscosity of the monoclonal antibody anti-IFNa to the greatest extent. However, a similar effect was observed with three other MAbs (Table 2). The buffer components and polypeptide concentration vary between different polypeptides, but the viscosity reducing effect of DMSO and DMA on each individual solution is obvious. In fact, a 2-3 fold decrease in solution viscosity was observed in some instances (Table 2). 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Conc. 
                   
                 Δη DMSO   
                 Δη DMA   
                   
                   
               
               
                 Polypeptide 
                 (mg/mL) 
                 Formulation 
                 (cP) 
                 (cP) 
                 F DMSO   
                 F DMA   
               
               
                   
               
             
            
               
                 Anti-IFNa 
                 150-200 
                 25, 50 and 75 mM 
                 see FIGS. 
                 see FIGS. 
                 2-3 
                 2-3 
               
               
                   
                   
                 HisCl, pH 5.2-6.5, 
                 1-3 
                 1-3 
               
               
                   
                   
                 ±co-solvent 
               
               
                 Anti-IFNa 
                 150 
                 25, 50 and 75 mM 
                 see FIG. 3 
                 see FIG. 3. 
                 1.5 
                 1.5 
               
               
                   
                   
                 HisCl, Arginine 
               
               
                   
                   
                 Chloride, ±co- 
               
               
                   
                   
                 solven 
               
               
                 Anti-IL17 
                 170 
                 30 mM HisCl, pH 
                 14 
                 23 
                 1.5 
                 2   
               
               
                   
                   
                 5.5, ±3% co- 
               
               
                   
                   
                 solven 
               
               
                 anti-CD20 
                 170 
                 20 mM sodium 
                  8 
                  5 
                 1.4 
                 1.2 
               
               
                   
                   
                 acetate, 4% 
               
               
                   
                   
                 trehalose, 
               
               
                   
                   
                 0.02% PS20, pH 
               
               
                   
                   
                 5.3, ±3% co- 
               
               
                   
                   
                 solven 
               
               
                 Anti-Beta 7 
                 200 
                 20 mM HisCl, pH 
                 20 
                 NA 
                 1.2-1.5 
                 NA 
               
               
                   
                   
                 5.8, ±5% DMSO 
               
               
                   
               
               
                 Δη DMSO/DMA  is the change in shear viscosity when DMSO or DMA is added. 
               
               
                 F DMSO/DMSO  is the fold change in shear viscosity or the ratio of the buffer viscosity without co-solvent to buffer viscosity with co-solvent. 
               
            
           
         
       
     
     To further investigate the viscosity reducing properties of the polar solvents DMSO and DMA, the solution pH was varied from 5.2 to 6.5 and shear viscosity measured. The solution viscosity was reduced by DMSO at all solution pH values tested ( FIG. 2 ). Clearly, the observed effect is a direct result of the polar additive DMSO. Indeed, solution pH dramatically affects solution viscosity in the absence of DMSO and the addition of DMSO attenuates the pH dependent viscosity changes ( FIG. 2 ). 
     Another common excipient for solubility enhancement and viscosity reduction of polypeptide solutions is arginine chloride. The effect of DMSO and DMA was investigated in the presence of varying amounts of arginine chloride to determine if these polar solvents exhibit further viscosity reducing effects. Herein, the addition of DMSO and DMA to polypeptide solutions is shown to further reduce solution viscosity in the presence of arginine chloride ( FIG. 3 ). 
     Herein, the effects of dimethyl sulfoxide and dimethyl acetamide have been explored. Clearly, these polar constituents decrease solution viscosity of high concentration polypeptide therapeutics. DMO and DMA may be used to increase the manufacturability and delivery of high concentration polypeptide formulations.