HK1164151A - High concentration antibody and protein formulations - Google Patents

Description

High concentration antibody and protein formulations

The present application is a divisional application of the invention application having an application date of 2004, 3/29, and a chinese application No. 200480015556.2, entitled "high concentration antibody and protein preparation".

Background

Technical Field

The present invention relates to highly concentrated antibody formulations particularly suitable for subcutaneous administration. The invention further provides stable, highly concentrated (e.g.. gtoreq.100 mg/ml protein) liquid formulations.

Description of the Related Art

There is a significant need for highly concentrated liquid antibody formulations. However, highly concentrated protein formulations encounter several difficulties. One problem is instability due to particulate formation (particulate). The use of reconstituted lyophilized products to produce liquid formulations has solved this problem by using surfactants (e.g., polysorbates), but surfactants are not suitable for liquid formulations because they present difficulties for further processing. In addition, surfactants also do not reduce the increased viscosity that results from the large number of intermolecular interactions that are brought about by the polymeric nature of the antibodies.

Although surfactants have been shown to significantly reduce the extent of protein microparticle formation, they do not address the problem of increased viscosity, which makes handling and application of concentrated antibody formulations difficult. Due to the high molecular nature and potential intermolecular interactions of antibodies, antibodies tend to form viscous solutions at high concentrations. In addition, pharmaceutically acceptable sugars are often used in large amounts as stabilizers. Such sugars may enhance intermolecular interactions, thereby increasing the viscosity of the formulation. Highly viscous formulations are difficult to manufacture, difficult to inhale into a syringe, and difficult to inject subcutaneously. The use of pressure in handling viscous formulations causes excessive foaming, which can result in denaturation and inactivation of the active bioproduct. This problem also lacks a satisfactory solution.

Although the prior art indicates numerous examples of excipients that can be suitably employed in the manufacture of pharmaceutical formulations, few proteins have been successfully formulated at concentrations in excess of 100mg/ml, and few have described techniques for formulating proteins at concentrations in excess of 100 mg/ml.

Applicants have found that arginine, and in particular arginine-HCl, is particularly suitable for highly concentrated liquid protein or antibody formulations.

Stable isotonic lyophilized protein formulations are disclosed in PCT publication WO97/04801, published 1997, 2/13, the entire disclosure of which is incorporated herein by reference. The disclosed lyophilized formulations can be reconstituted to produce liquid formulations of high protein concentration without significant loss of stability. However, the potential problems associated with the high viscosity of reconstituted formulations have not been addressed. Protein aggregation has been previously reduced by the addition of sugars, but doing so can significantly increase viscosity and osmotic pressure, resulting in impractical processing and use.

The applicants PCT application, WO02/30463, published on 4/18/2002, discloses high protein concentration but low viscosity formulations by (1) low pH (about 4.0 to 5.3); (2) high pH (about 6.5 to 12.0) or (3) is achieved by increasing the total ionic strength of the formulation by adding salts or buffers. However, when increased ionic strength reduces the viscosity of the formulation (e.g., with NaCl), it can also result in increased solution turbidity, which is often associated with the formation of protein particles (e.g., aggregation). Therefore, optimal high concentration protein formulations must overcome the difficulties of stability, viscosity, osmotic pressure and haziness.

Summary of The Invention

The present invention relates to highly concentrated protein or antibody formulations that are stable and are of low viscosity and low turbidity.

In particular, the present invention relates to a highly concentrated antibody formulation of low turbidity comprising a protein or antibody (100-260mg/ml), histidine (10-100mM), arginine-HCl (50-200mM) and polysorbate (0.01% -0.1%), having a pH of 5.5-7.0, a viscosity of 50cs or less and an osmolality of 200mOsm/kg-450 mOsm/kg. Alternatively, the protein or antibody in the formulation may be in the range of 120-260mg/ml, alternatively 150-260mg/ml, alternatively 180-260mg/ml, alternatively 200-260 mg/ml. Or the osmolality is in the range of 250-350 mOsm/kg. Alternatively, the arginine-HCl concentration is in the range of 100-.

Alternatively, the invention relates to a highly concentrated antibody formulation of low turbidity comprising an antibody (40-150mg/ml), histidine (10-100mM), a sugar (e.g., trehalose or sucrose, 20-350mM) and polysorbate (0.01% -0.1%).

In a particular embodiment, the invention provides a formulation comprising a high concentration of large molecular weight proteins, such as antibodies or immunoglobulins. The antibody may be, for example, an antibody directed against a particular predetermined antigen. In a particular aspect, the antigen is IgE (e.g., rhuMAbE-25 and rhuMAbE-26 described in U.S. Pat. No. 6,329,509 and WO 99/01556). Alternatively, the anti-IgE antibody may be a peptide found in corn et al, j.clin.invest.99 (5): 879-887(1997), CGP-5101(Hu-901) described in W092/17207, and ATTC accession Nos. BRL-10706 and 11130, 11131, 11132, 11133. Alternatively, antigens may include: CD proteins CD3, CD4, CD8, CD19, CD20, CD34, and CD 40; a member of the HER receptor family such as the EGF receptor, the HER2, HER3 or HER4 receptor; 2C4, 4D5, PSCA, LDP-2, cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, and α v/β 3 integrins including the α -and β -subunits thereof (e.g., anti-CD 11a, anti-CD 18, or anti-CD 11b antibodies); growth factors such as VEGF; blood group antigens; flk2/flt3 receptor; obesity (OB) receptors; mpl receptor, CTLA-4 and protein C.

The formulation of the invention may be a pharmaceutical formulation. In a particular aspect, the formulation is administered subcutaneously.

In yet another embodiment, the invention provides a method of treating, preventing or treating a disease treatable with a formulated protein or antibody, comprising administering a formulation disclosed herein comprising a therapeutically effective amount of a protein or antibody. Such formulations are particularly useful for subcutaneous administration. In one aspect, the disease is an IgE-mediated disease. Still further, the IgE-mediated diseases are allergic rhinitis, asthma (e.g., allergic asthma and non-allergic asthma), atopic dermatitis, allergic gastrointestinal disease, hypersensitivity reactions (e.g., anaphylaxis, urticaria, food allergy, etc.), allergic bronchopulmonary aspergillosis, parasitic diseases, interstitial cystitis, hyper IgE syndrome, ataxia telangiectasia, Wiskott-Aldrich syndrome, thymic lymphodysplasia, IgE myeloma, and graft-versus-host reaction.

In one embodiment, the present invention provides an article of manufacture comprising a container containing a formulation as disclosed herein. In one aspect, the article is a pre-filled syringe. In another aspect, the pre-filled syringe is further included in an injection device. In another aspect, the injection device is an auto-injector.

The present application relates to the following.

1. A stable, low turbidity, liquid formulation comprising (a) a protein or antibody in an amount of 100 to 260mg/ml, (b) arginine-HCl in an amount of 50 to 200mM, (c) histidine in an amount of 10 to 100mM, (d) polysorbate in an amount of 0.01 to 0.1%, wherein the formulation further has a pH of from 5.5 to 7.0, a kinematic viscosity of about 50cs or less, and an osmolality of from 200 to 450 mOsm/kg.

2. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 120mg/ml to 260 mg/ml.

3. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 150mg/ml to 260 mg/ml.

4. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 180mg/ml to 260 mg/ml.

5. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 200mg/ml to 260 mg/ml.

6. The formulation of item 1, wherein the protein or antibody is at a concentration of about 150 mg/ml.

7. The formulation of item 1, wherein the osmolality ranges from 250 to 350 mOsm/kg.

8. The formulation of item 1, wherein the arginine-HCl concentration ranges from 100mg/ml to 200 mg/ml.

9. A stable, low turbidity liquid formulation comprising (a) an anti-IgE monoclonal antibody in an amount of 100 to 260mg/ml, (b) arginine-HCl in an amount of 50 to 200mM, (c) histidine in an amount of 10 to 100mM, (d) polysorbate in an amount of 0.01 to 0.1%, wherein the formulation further has a pH value of from 5.5 to 7.0, a kinematic viscosity of about 50cs or less, and an osmolality of from 200 to 450 mOsm/kg.

10. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 120mg/ml to 260 mg/ml.

11. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 150mg/ml to 260 mg/ml.

12. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 180mg/ml to 260 mg/ml.

13. The formulation of item 1, wherein the concentration of the protein or antibody ranges from 200mg/ml to 260 mg/ml.

14. The formulation of item 1, wherein the protein or antibody is at a concentration of about 150 mg/ml.

15. The formulation of item 1, wherein the osmolality ranges from 250 to 350 mOsm/kg.

16. The formulation of item 1, wherein the anti-IgE antibody is selected from rhuMAbE25, rhuMAbE26, or Hu-901.

17. The formulation of item 1, wherein the anti-IgE antibody is rhuMAbE 25.

18. The formulation of item 1, wherein the anti-IgE antibody is rhuMAbE 26.

19. The formulation of item 1, wherein the anti-IgE antibody is Hu-901.

20. A stable, low turbidity liquid formulation comprising (a) an anti-IgE antibody in an amount of about 150mg/ml, (b) arginine-HCl in an amount of 200mM, (c) histidine in an amount of 20mM, (d) polysorbate in an amount of 0.02%, wherein the formulation further has a pH of 6.0.

21. The formulation of item 20, wherein the anti-IgE antibody is E25.

22. An article of manufacture comprising a container containing the formulation of item 1.

23. The article of item 22, wherein the container is a syringe.

24. The article of manufacture of item 23, wherein the syringe is further contained in an injection device.

25. The article of claim 24, wherein the injection device is an auto-injector.

26. The formulation of item 1, wherein the formulation is reconstituted.

27. The formulation of clause 26, wherein the concentration of the protein or antibody in the reconstituted formulation is about 2-40 times higher than the concentration prior to lyophilization.

28. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20.

29. The method of item 28, wherein the IgE-mediated disease is selected from allergic rhinitis, asthma, allergic asthma, non-allergic asthma, atopic dermatitis, or gastrointestinal disease.

30. The method of item 28, wherein the IgE-mediated disorder is allergic rhinitis.

31. The method of item 28, wherein the IgE-mediated disease is allergic asthma.

32. The method of item 28, wherein the IgE-mediated disease is asthma.

33. The method of item 28, wherein the IgE-mediated disease is atopic dermatitis.

34. The method of item 28, wherein the IgE-mediated disease is selected from hypersensitivity, allergic bronchopulmonary aspergillosis, parasitic disease, interstitial cystitis, hyper-IgE syndrome, ataxia telangiectasia, Wiskott-Akdrich syndrome, thymic lymphodysplasia, IgE myeloma, or graft-versus-host reaction.

35. The method of item 28, wherein the IgE-mediated disease is a hypersensitivity reaction.

36. The method of item 35, wherein the hypersensitivity disorder is selected from the group consisting of anaphylaxis, urticaria, or food allergy.

37. The method of item 36, wherein the hypersensitivity disorder is food allergy.

38. The method of item 37, wherein the food allergy is caused by exposure to legumes.

39. The method of item 38, wherein the legume is peanut.

40. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof an effective amount of the formulation of item 20 in combination with an antihistamine.

41. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of an antihistamine.

42. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with a bronchodilator.

43. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of a bronchodilator.

44. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with a glucocorticoid.

45. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of a glucocorticoid.

46. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with a glucocorticoid.

47. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of a glucocorticoid.

48. A method of treating an IgE-mediated disease comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of allergen desensitization therapy.

49. A method of treating an IgE-mediated disease comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with an NSAID.

50. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of item 20 in combination with the administration of an NSAID.

Drawings

FIG. 1 hydrophobic interaction chromatography of pepsin digested anti-IgE monoclonal antibodies. Samples were prepared at different pH values and buffers: (●)20mM acetate, (. DELTA.) 20mM succinate, (. tangle-solidup.) 20mM Na₂HPO₄，(Λ)20mM K₂PO₄And (. about.20 mM Tris buffer. The samples were stored at 30 ℃ for 6 months.

FIG. 2 size exclusion chromatography of anti-IgE monoclonal antibodies stored at 40 ℃ for 6 months. Samples were prepared at different pH values and buffers: (■)20mM glutamate, (●)20mM acetate, (. DELTA.) 20mM succinate, (□)20mM histidine, (. tangle-solidup.) 20mM Na₂HPO₄，20mMK₂PO₄And (. about.20 mM Tris buffer.

FIG. 3. Activity of anti-IgE monoclonal antibodies stored at 30 ℃ for 6 months. Samples were prepared at different pH values and buffers: (●)20mM acetate, (. DELTA.) 20mM succinate, (□)20mM histidine, (. tangle-solidup.) 20mM Na₂HPO₄，20mM K₂PO₄And (. about.20 mM Tris buffer.

FIG. 4. Effect of Polysorbate 20 on turbidity of stressed anti-IgE monoclonal antibodies. The sample contained 100mg/ml antibody, 20mM succinate, 192mM trehalose and respective amounts of polysorbate 20, pH 6.0. The polysorbate concentration was (■)0, (. tangle-solidup.) 0.01%, (●) 0.02% and (. DELTA.) 0.05%.

Figure 5 anti IgE monoclonal antibody at-150 mg/ml and different excipient (a-solidup) CaCl₂，MgCl₂And (. DELTA.) the turbidity of arginine-HCl.

FIG. 6. turbidity of anti-IgE monoclonal antibodies at-150 mg/ml with various excipients. Samples were stored at (. tangle-solidup.) to 70 ℃, (■)2 to 8 ℃, (. DELTA.) 15 ℃, (□)30 ℃ and40℃。

FIG. 7 hydrophobic interaction chromatography analysis of papain digested anti-IgE monoclonal antibodies. Samples are prepared with various excipients in a ratio of 150mg/ml and stored in-70 ℃ (■)2-8 ℃ (a tangle-solidup) 15 ℃ (Δ)30 ℃ and (□)40 ℃.

FIG. 8 anti-IgE monoclonal antibodies at-150 mg/ml in (■)200mM arginine-HCl, 23mM histidine, pH6.0, (. tangle-solidup.) 182mM arginine-HCl, 20mM histidine, pH6.0, (●)182mM arginine-HCl, 20mM histidine, 91mM sucrose, pH6.0, (□)50mM MgCl₂27mg/ml trehalose, 0.01% acetate, (. DELTA.) 50mM MgCl₂、30mM MgAc₂0.01% acetate, and (. smallcircle.) 50mM MgCl₂、45mM MgAc₂Size exclusion chromatography in 0.01% acetate. The samples were stored at 30 ℃ for 6 months.

FIG. 9 hydrophobic interaction chromatography analysis of papain digested anti-IgE monoclonal antibodies. The samples shown are formulated (■) at 200mM arginine-HCl, 23mM histidine, (. tangle-solidup.) 182mM arginine-HCl, 20mM histidine, (●)182mM arginine-HCl, 20mM histidine, 91mM sucrose, (□)50mM MgCl₂27mg/ml trehalose, 0.01% acetate, (. DELTA.) 50mM MgCl₂、30mM MgAc₂0.01% acetate, and (. smallcircle.) 50mM MgCl₂、45mM MgAc₂0.01% acetate. The samples were stored at 30 ℃ for 6 months.

Figures 10A and 10b show a comparison of the full length sequences of the variable and constant chains of anti-IgE antibodies E25, E26 and Hu-901. The CDR regions of Hu-901 are underlined. For E25 and E26, Chothia defined CDR regions are indicated in bold type, and Kabat defined CDR regions are boxed. FIG. 10A shows the light chain sequences of E25, E26, and Hu-901 (SEQ ID NOS: 1-3), while FIG. 10B shows the heavy chain sequences of E25, E26, and Hu-901 (SEQ ID NOS: 4-6).

Detailed description of the preferred embodiments

I. Definition of

"protein" refers to an amino acid sequence having a chain length sufficient to produce high levels of tertiary and/or quaternary structure. This is in contrast to "polypeptides" or other small molecular weight drugs that do not have such a structure. Generally, the molecular weight of the proteins herein is at least about 15 to about 20kD, preferably at least about 20 kD.

Examples of proteins encompassed by the definition herein are mammalian proteins, e.g., growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; a thyrotropin; a lipoprotein; alpha-1-antitrypsin; an insulin a chain; insulin B chain; proinsulin; a follicle stimulating hormone; a calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor and von willebrand factor; anti-coagulation factors such as protein C; atrial natriuretic peptides; a pulmonary surfactant; a plasminogen kinase, such as urokinase or tissue-type plasminogen kinase (t-PA); bombesin; thrombin; tumor necrosis factor-alpha and-beta; enkephalinase; the chemokine RANTES (which is regulatable upon activation and is usually expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; (ii) a muller inhibitor; a relaxin a chain; a relaxin B chain; (ii) prorelaxin; a murine gonadotropin-associated peptide; deoxyribonuclease; a statin; an activin; vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors; an integrin; protein A or D; rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophin-3, -4, -5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGN-beta; platelet Derived Growth Factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4 or TGF-beta 5; insulin-like growth factors-I and-II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I); insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD19, and CD 20; erythropoietin (EPO); platelet auxin (TPO); osteogenic inducing factors (osteoinductive factors); an immunotoxin; bone Morphogenetic Protein (BMP); interferons, such as interferon- α, - β, and- γ; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 through IL-10; superoxide dismutase; a T cell receptor; membrane surface proteins; decay Acceleration Factor (DAF); partial coatings of viral antigens, such as AIDS; a transporter protein; a homing receptor; an address element; a regulatory protein; an immunoadhesin; an antibody; and biologically active fragments or variants of any of the foregoing polypeptides.

The protein of the formulation is preferably substantially pure, and preferably substantially homogeneous (i.e., free of contaminating proteins, etc.). By "substantially pure" protein is meant that the component comprises at least about 90% by weight protein (based on the total weight of the component), preferably at least about 95% by weight protein. By "substantially homogeneous" protein is meant that the component comprises at least 99% by weight protein, based on the total weight of the component.

In some examples, the protein is an antibody. For example, an antibody can bind to any of the molecules described above. Typical target molecules for antibodies of the invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD 40; a member of the HER receptor family, such as the EGF receptor, HER2, HER3 or HER4 receptor; 2c4, 4D5, PSCA, LDP-2; cell adhesion molecules, such as LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, and α v/β 3 integrin, including its α or β subunit (e.g., anti-CD 11a, anti-CD 18, or anti-CD 11b antibody); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; a lipid (OB) receptor; an mpl receptor; CTLA-4, protein C, and the like.

The term used therein "Antibodies "include monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies (diabodies), and single chain molecules), and antibody fragments (e.g., Fab, F (ab')₂And Fv). The term "immunoglobulin" (Ig) is used herein interchangeably with "antibody".

The basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 elementary heterotetramer units with an additional polypeptide called the J chain, containing 10 antigen binding sites, while IgA antibodies contain 2-5 elementary 4-chain units, the elementary 4-chain units being capable of co-polymerizing with the J chain to form multivalent aggregates. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable region (V) at the N-terminus_H) Followed by three constant regions (C) for each alpha and gamma chain_H) For mu and epsilon isoforms followed by four C_HAnd (4) a zone. Each L chain has a variable region (V) at the N-terminus_L) Followed at the other end by a constant region. The constant region of the light chain corresponds to the first constant region of the heavy chain (align), while the variable region of the light chain corresponds to the variable region of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable regions. V_HAnd V_LTogether, form a single antigen binding site. For the structure and properties of different classes of antibodies see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (eds), Appleton&Lange, Norwalk, CT, 1994, page 71 and chapter 6.

L chains from any vertebrate species can be assigned to one of two distinctly different types, called kappa and lambda, based on the amino acid sequence of their constant regions. Depending on the amino acid sequence of their heavy Chain (CH) constant regions, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively. The γ and μ classes are further divided into subclasses according to the relatively small differences in the sequence and function of CH, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

The term "variable" refers to the fact that certain fragments of the variable region differ greatly in sequence between antibodies. The V regions mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable region. Rather, the V region consists of a relatively invariant region, called the Framework Region (FR), of about 15-30 amino acid residues, separated by several shorter regions, called the "hypervariable region" or sometimes the "complementarity determining region" (CDR), with extreme variability, of about 9-12 amino acid residues each in length. The variable regions of each native heavy and light chain comprise four FRs, mostly in a β -sheet structure, 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, together with hypervariable regions from the other chain, forming the antigen-binding site of an antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" (also referred to as "complementarity determining regions" or CDRs) when used herein refers to antibody amino acid residues (usually three or four short regions of extreme sequence variability) located within immunoglobulin V region domains that form the antigen-binding site and are the primary determinants of antigen specificity. There are at least two methods to identify CDR residues: (1) according to the method of cross-species sequence variability (i.e.,kabat et al, Sequences of Proteins of Immunological Interest (National institute of Health, Bethesda, MS 1991)); and (2) methods based on crystallization studies of antigen-antibody complexes (Chothia, c. et al, j.mol.biol.196: 901-917(1987)). In this sense, however, the two residue identification techniques determine overlapping regions, rather than identical regions, which can be combined to determine hybrid CDRs.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprise the same population except for a small number of possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, each monoclonal antibody is directed against only a single determinant of the antigen, unlike conventional (polyclonal) antibody preparations, i.e. typically different antibodies are directed against different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage that they are synthesized using hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" refers to the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method described by Kohler et al (Nature, 256: 495(1975)), or by recombinant DNA techniques (see U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al (Nature, 352: 624-.

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, and the remainder of the chain 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;)Etc., Proc. Natl. Acad. Sci. USA,81: 6851-6855(1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable region antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

An "intact" antibody is one which comprises an antigen binding site and a CL and at least a heavy chain region C_H1、C_H2 and C_H3. The constant region can be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" includes a portion of an intact antibody, preferably including the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a bifunctional antibody; linear antibodies (see U.S. Pat. No.5,641,870, example 2; Zapata et al, Protein Eng.8(10)：1057-1062[1995]) (ii) a Single chain antibody molecules and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, which is a designation reflecting the ability to crystallize readily. The Fab fragment consists of the entire L chain as well as the variable region of the H chain (V)_H) And the first constant region (C) of a heavy chain_H1) And (4) forming. Each Fab fragment is monovalent for antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of the antibody produced a single large F (ab')₂Fragments which correspond roughly to two disulfide-linked Fab fragments with different antigen-binding activities and which still have the ability to crosslink antigen. Fab 'fragments differ from Fab fragments, Fab' at C_HRegion 1 has several additional residues at the carboxy terminus, including one or more cysteines from the hinge region of the antibody. Fab '-SH is understood here to mean Fab' which carries a free thiol group at a cysteine residue in the constant region. F (ab')₂Generation of antibody fragments originally as Fab' fragmentsThe counterpart was produced with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy terminal portions of two H chains linked by a disulfide bond. The effector function of an antibody is determined by the sequence of the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This fragment consists of a dimer of a heavy chain variable region and a light chain variable region, tightly non-covalently linked. In this conformation, the three hypervariable regions of each variable region interact with each other, at V_H-V_LThe dimeric surface defines an antigen binding site. Collectively, these six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable region (or F)_VThe half of the above containing only two antigen-specific hypervariable regions) also have the ability to recognize and bind antigen, but with a lower affinity compared to the complete binding site.

"Single chain Fv" also abbreviated as "sFv" or "scFv" antibody fragments, including V of antibodies_HAnd V_LDomains, which domains are present on a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LThe structural domains also contain a polypeptide linker which enables the sFv to form the structure required for antigen binding. For an overview of sFvs, see Pluckthun in "pharmacology of monoclonal antibodies", Vol.113, eds Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994).

The term "bifunctional antibody" refers to small antibody fragments, obtained by the use of V_HAnd V_LShort linkers between the regions (about residues 5-10) construct sFv fragments (see preceding paragraph), thus making pairs of V regions between chains, rather than within chains, resulting in bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" sFv fragments in which the V of both antibodies_HAnd V_LThe regions exist differentlyOn the polypeptide chain of (a). Bifunctional antibodies are described, for example, in EP404,097; WO 93/11161; hollinger et al, Proc. Natl. Acad. Sci. USA90: 6444-.

An antibody that "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to the particular polypeptide or epitope on the particular polypeptide, substantially without binding to any other polypeptide or polypeptide epitope.

The term "solid phase" describes an anhydrous matrix to which an antibody of the invention can be attached. Examples of solid phases included herein include those formed partially or entirely of glass (e.g., glass to control pore size), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone. In certain embodiments, depending on the particular situation, the solid phase may comprise the wells of an assay plate; in other embodiments, it is a purification column (e.g., an affinity chromatography column). This term also includes the separate solid phases of the whole article, such as those described in U.S. Pat. No.4,275,149.

"humanized" antibodies of non-human origin (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains, or fragments comprising minimal sequence derived from non-human immunoglobulins (e.g., Fv, Fab ', F (ab') 2, or other antigen-binding antibody sequences). Humanized antibodies are mostly immunoglobulins of the human type (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient are replaced by CDR residues from an antibody of the non-human type, e.g. a murine, rat or rabbit antibody (donor antibody), of the desired specificity, affinity and capacity. In some instances, Fv Framework Region (FR) residues of a human immunoglobulin are substituted for corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in either the acceptor antibody or the imported CDR or framework sequences. These modifications can further refine and optimize the performance of the antibody. The humanized antibody will also most desirably comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Details of the inventionSee Jones et al, Nature, 321: 522-525 (1986); reichmann et al, Nature,332: 323-329 (1988); and Presta, curr, op, struct, biol,2：593-596(1992)。

a "species-dependent antibody", e.g., a mammalian anti-human IgE antibody, is an antibody that has a greater binding affinity for an antigen from a first mammalian species than for a homolog of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (i.e., has no more than about 1 × 10)^-7Binding affinity (Kd) value of M, or not more than 1X 10^-8M, or not more than 1X 10^-9M) but has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50-fold, at least about 500-fold, or at least about 1000-fold weaker than its affinity for the non-human antigen. The species-dependent antibody may be any of the various types of antibodies as defined above, but is preferably a humanized or human antibody.

Antibody "effector functions" refer to those biological activities that are due to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), which 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 (e.g., B cell receptors); and B cell activation.

"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 antibodies that bind to a target cell, which subsequently causes lysis of the target cell. The major cells mediating ADCC express only Fc γ RIII in NK cells, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Summary of FcR expression on hematopoietic cells is found in ravatch and Kinet, annu. TABLE III on page 464 of 457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, for example as described in us patent 5500362 or 5821337. Potent effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively (or additionally), the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model as described by Clynes et al (PNAS (USA), 95: 652-.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc portion of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is a receptor (gamma receptor) that binds an IgG antibody, including the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses (including allelic variants and selective splice forms of these receptors). Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor") having similar amino acid sequences, which differ primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (see review M.in Daeron, Annu. Rev. Immunol.15: 203-234 (1997)). FcR is reviewed in ravatch and Kinet, annu.rev.immunol.9: 457-92 (1991); capel et al, immunological methods 4: 25-34 (1994); and de Haas et al, j.lab.clin.med.126: 330-41(1995). The term "FcR" herein covers other fcrs, including those to be identified in the future. The term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al, J Immunol 117: 587(1976) and Kim et al, J Immunol 24: 249 (1994)).

A "human effector cell" is a leukocyte that expresses one or more fcrs and performs effector functions. Preferably the cells express at least Fc γ RIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMC and NK cells are preferred. The effector cells may be isolated from their natural source, e.g., from blood or PBMCs as described herein.

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. Binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that binds to a cognate antigen initiates the complement activation pathway. To evaluate complement activation, CDC assays can be performed, as described by Gazzano-Santoro et al (J. Immunol. methods 202: 163 (1996)).

An "isolated" polypeptide refers to a polypeptide that has been identified and isolated and/or harvested from its natural environmental component. Contaminant components of the natural environment of the peptide are those that would interfere with the use of the polypeptide for diagnostic and therapeutic purposes and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is purified as: (1) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotor-type protein sequencer, or (2) to the extent that SDS-PAGE is electrophoretically homogeneous under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. An isolated polypeptide includes a polypeptide in situ within a recombinant cell because at least one component of the polypeptide's natural environment is not present. However, at least one purification step is typically used to prepare the isolated polypeptide.

An "isolated" nucleic acid molecule encoding a polypeptide and antibody herein is a nucleic acid molecule that is identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, the isolated nucleic acid is free from association with all components associated with the environment in which it is produced. Isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form that is different from the form or environment in which they are found in nature. An isolated nucleic acid molecule therefore differs from a nucleic acid encoding a polypeptide or antibody herein that naturally occurs in a cell.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" to another nucleic acid when the nucleic acid is in a functionally relevant position to the other nucleic acid sequence. For example, if a polypeptide is expressed as a precursor protein involved in the secretion of the polypeptide, DNA for the presequence or secretory leader is operably linked to DNA for the polypeptide; a promoter or enhancer may be operably linked to a coding sequence when it affects the transcription of the coding sequence; or a ribosome binding site can be operably linked to a coding sequence when it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading order. However, enhancers need not be contiguous. Ligation may be achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers can be used according to conventional practice.

The term "tagged epitope (epitope tagged)" when used herein refers to a chimeric polypeptide comprising a polypeptide or antibody described herein fused to a "tag polypeptide". The marker polypeptide has sufficient residues to provide an epitope against which antibodies can be raised, yet is short enough so as not to interfere with the activity of the polypeptide to which it is fused. The marker polypeptide is preferably also sufficiently unique that the antibody does not substantially cross-react with other epitopes. Suitable marker polypeptides typically have at least six amino acid residues, usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

The term "immunoadhesin" is defined herein as an antibody-like molecule that combines the binding domain of a heterologous "adhesin" protein (e.g., a receptor, ligand, or enzyme) with an immunoglobulin constant region. Structurally, immunoadhesins comprise the fusion of the amino acid residues of an adhesin having the desired binding specificity, which amino acid residues are distinct from the antigen recognition and binding site (antigen binding site) of an antibody (i.e., "heterologous"), with immunoglobulin constant region sequences. The adhesin part of an immunoadhesion molecule is typically a contiguous amino acid sequence comprising at least one binding site for a receptor or ligand. The immunoglobulin constant region sequences in immunoadhesins can be derived 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. Ig fusions preferably include the replacement of at least one variable region within an Ig molecule with a region of a polypeptide or antibody described herein. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. See also U.S. Pat. No.5,428,130 issued on month 6 and 27 of 1995 for the production of immunoglobulin fusions.

A "stable" formulation is one in which the protein therein substantially maintains its physical and chemical stability and integrity during storage. Various analytical techniques exist in the art for measuring Protein stability, as described in Peptide and Protein Drug Delivery, 247-: 29-90 (1993). The stability over a selected period of time may be measured at a selected temperature. For rapid screening, the formulations can be maintained at 40 ℃ for 2 weeks to 1 month, at which time stability is measured. When the formulation is to be stored at 2-8 ℃, typically the formulation should be stable at 30 ℃ or 40 ℃ for at least 1 month and/or at 2-8 ℃ for at least 2 years. When the formulation is to be stored at 30 ℃, typically the formulation should be stable for at least 2 years at 30 ℃ and/or for at least 6 months at 40 ℃. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which less than about 10%, and preferably less than about 5%, of the protein is present as aggregates in the formulation. In another embodiment, any increase in polymer formation during storage of the formulation may be determined.

A "reconstituted" formulation is one that has been prepared by dissolving a lyophilized protein or antibody formulation in a diluent to have the protein distributed throughout. The reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient in need of treatment with the protein of interest, and in certain embodiments of the invention, may be a formulation suitable for subcutaneous administration.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of from about 250 to 350 mOsm. The term "hypotonic" describes a formulation having an osmotic pressure lower than that of human blood. Accordingly, the term "hypertonic" is used to describe a formulation having an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or freezing type osmometer. The formulations of the present invention are hypertonic as a result of the addition of salts and/or buffers.

"pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic in the concentrations and manner in which they are formulated. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, sulfuric acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include straight and branched chain hydrocarbyl, aromatic, cyclic, cycloaliphatic, araliphatic, heterocyclic, saturated, unsaturated, monocarboxylic, dicarboxylic, tricarboxylic acids, including, for example, formic acid, acetic acid, 2-glycolic acid, trifluoroacetic acid, phenylacetic acid, trimethylacetic acid, t-butylacetic acid, anthranilic acid, propionic acid, 2-hydroxypropionic acid, 2-carbonylpropionic acid, malonic acid, cyclopentanepropionic acid (cyclopentaneppionic acid), cyclopentanepropionic acid (cyclopropanepropionic acid), 3-phenylpropionic acid, butyric acid, succinic acid, benzoic acid, 3- (4-hydroxybenzyl) benzoic acid, 2-acetoxy-benzoic acid, ascorbic acid, cinnamic acid, dodecylsulfuric acid, stearic acid, muconic acid, mandelic acid, succinic acid, embonic acid, fumaric acid, succinic acid, and mixtures thereof, Malic acid, maleic acid, hydroxymaleic acid, malonic acid, lactic acid, citric acid, tartaric acid, glycolic acid, saccharic acid, gluconic acid, pyruvic acid, glyoxylic acid, oxalic acid, methanesulfonic acid, succinic acid, salicylic acid, phthalic acid, palmitic acid (palmoic acid), palmeic acid, thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo (2, 2, 2) -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4' -methylenebis-3- (hydroxy-2-ene-1-carboxylic acid), hydroxynaphthoic acid.

"pharmaceutically acceptable bases" include inorganic and organic bases, which are non-toxic in the concentrations and manner in which they are formulated. For example, suitable bases include those formed from inorganic alkali metals, such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine, and organic non-toxic bases including primary, secondary and tertiary amines, substituted amines, cyclic amines and base ion exchange resins, [ e.g., N (R') 4⁺(wherein R' is independently H or C_1-4Hydrocarbyl radicals, e.g. ammonium, Tris)]For example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline and caffeine.

Additional pharmaceutically acceptable acids and bases useful in the present invention include those derived from amino acids such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

"pharmaceutically acceptable" buffers and salts include those derived from the acid and base addition salts specified above. Specific buffers and/or salts include histidine, succinate and acetate.

A "lyoprotectant" is a molecule that reacts with a protein of interestThe chemical and/or physical instability of the protein is effectively prevented or reduced during lyophilization and subsequent storage. Exemplary lyoprotectants include sugars and their corresponding sugar alcohols; amino acids, such as monosodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as ternary or higher molecular weight sugar alcohols, e.g. glycerol (glycerol), dextran, erythritol, glycerol (glycerol), arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol;and combinations thereof. Additional exemplary lyoprotectants include glycerol and gelatin, and melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include glycosides of non-reducing polyols selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, in particular those obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The pendant glycoside groups may be glucosidic or galactoside. Further examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred lyoprotectants are the non-reducing sugars trehalose or sucrose.

Adding a lyoprotectant to a pre-lyophilized formulation in a "lyoprotecting amount" means that the protein substantially retains its physical and chemical stability and integrity during lyophilization and storage after lyophilization of the protein in the presence of the lyoprotecting amount of lyoprotectant.

In preparing the reduced viscosity formulations of the present invention, care needs to be taken to utilize the above enumerated excipients and other additives, particularly when added at high concentrations, so as not to increase the viscosity of the formulation.

A "pharmaceutically acceptable sugar" is a molecule that, when combined with a protein of interest,chemical and/or physical instability of the protein during storage is effectively prevented or reduced. A "pharmaceutically acceptable sugar" may also serve as a "lyoprotectant" when the formulation is to be lyophilized and then reconstituted. Exemplary sugars and their corresponding sugar alcohols include: amino acids, such as monosodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as ternary or higher molecular weight sugar alcohols, e.g. glycerol (glycerol), dextran, erythritol, glycerol (glycerol), arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol;and combinations thereof. Additional exemplary lyoprotectants include glycerol and gelatin, and melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include glycosides of non-reducing polyols selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, in particular those obtained by reducing disaccharides such as lactose, maltose, lactulose and maltulose. The pendant glycoside groups may be glucosidic or galactoside. Further examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred pharmaceutically acceptable sugars are the non-reducing sugars trehalose or sucrose.

Adding a pharmaceutically acceptable sugar to a formulation (e.g., prior to lyophilization) in a "protective amount" means that the protein substantially retains its physical and chemical stability and integrity during storage (e.g., after reconstitution and storage).

A "diluent" of interest herein is an agent that is pharmaceutically acceptable (safe and non-toxic for administration to humans), useful for formulating liquid formulations, such as formulations that are reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In alternative embodiments, the diluent may comprise an aqueous solution of a salt and/or buffer.

A "preservative" is a compound that can be added to the formulations herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the manufacture of a multiple use (multi-dose) formulation. Examples of possible preservatives include octadecyl dimethyl phenyl ammonium chloride, hexa-hydrocarbyl quaternary ammonium chloride, benzalkonium chloride (a mixture of alkyl phenyl dimethyl ammonium chlorides, where the hydrocarbyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, hydrocarbyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred preservative herein is benzyl alcohol.

"treatment" refers to both therapeutic treatment as well as prophylactic or preventative measures. Subjects in need of medical treatment include those who have developed a disease and those in need of prevention of the disease.

By "mammal" for purposes of treatment is meant any animal classified as a mammal, including humans, domestic and farm animals, and zoo, competitive or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. Preferably, the mammal is a human.

A "disease" is any condition that would benefit from treatment with a protein. This includes chronic and acute diseases or conditions, including those pathological conditions that predispose a mammal to the disease. Non-limiting examples of diseases to be treated herein include cancer and allergy.

A "therapeutically effective amount" is at least the minimum concentration required to cause a measurable improvement or prevention of a particular disease. Knowing that a therapeutically effective amount of a protein is known in the art, an effective amount of a protein as disclosed hereinafter can be determined by standard techniques within the capabilities of the skilled artisan, e.g., an ordinary physician.

As used herein, "viscosity" may be "kinematic viscosity" or "absolute viscosity". "kinematic viscosity" is a measure of resistance to liquid flow under the influence of gravity. When two equal volumes of fluid are placed in the same capillary viscometer and allowed to gravity flow, a viscous fluid takes longer to flow through the capillary than a lower viscous fluid. If one fluid takes 200 seconds to complete its flow and the other 400 seconds, the second fluid is twice as viscous as the first on a kinematic viscosity scale. "absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density:

absolute viscosity (kinematic viscosity) x density

Kinematic viscosity has the unit L²Where L is the length and T is the time. Generally, kinematic viscosity is expressed in centistokes (cSt). The international standard unit for kinematic viscosity is mm²And/s is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The international standard unit of absolute viscosity is millipascal-seconds (mPa-s), 1cP being 1 mPa-s.

As used herein, an "antihistamine" is an agent that counters the physiological effects of histamine. Histamine and its receptor, H₁And H₂The combination produces characteristic allergic symptoms and effects, or itching, redness, swelling, etc. Many antihistamines work by blocking the binding of histamine to its receptors H1, H2; while others are believed to act by inhibiting the release of histamine. Examples of antihistamines are clofenamide, diphenhydramine, promethamine, cromolyn sodium, astemizole, pipoheptadine maleate, bronheniramine maleate, chlorpyrilamine maleate, cetirizine hydrochloride, clemastine fumarate, diphenylcycloheptidine hydrochloride, dexbrompheniramine maleate, dextrochlorpheniramine maleate, chlorophylline diphenhydramine hydrochloride, ducrehydramine succinate, fexofenadine hydrochloride, terphenyladine hydrochloride, hydroxyzine hydrochloride, loratidine hydrochloride, tripelennamine citrateTripheniramine hydrochloride and propirolidine hydrochloride.

As used herein, "bronchodilators" describe agents that counteract or reverse bronchoconstriction, a physiological event that typically occurs in an early phase asthmatic response and results in decreased lung capacity and wheezing. Examples of bronchodilators include the widely acting alpha and beta adrenergic adrenaline, beta adrenergic albuterol, pirbuterol, metaproterenol, salmeterol and neoproterenol. Bronchodilation can also be achieved by administration of xanthines, including aminophylline and theophylline.

As used herein, "glucocorticoids" describe steroid-based agents with anti-inflammatory activity. Glucocorticoids are commonly used to attenuate the late-stage asthmatic response. Examples of glucocorticoids include, dehydrocortisone, beclomethasone dipropionate, triamcinolone acetonide, flunisolide, betamethasone, budesonide, dexamethasone, fludrocortisone acetate, flunisolide, fluticasone propionate, hydrocortisone, methylprednisolone, prednisolone, dehydrocortisone, and triamcinolone.

As used herein, "non-steroidal anti-inflammatory drug" or "NSAID" describes non-steroid based agents having anti-inflammatory activity. Examples of NSAIDs include acetaminophen, acetylsalicylic acid, sodium bromfenac, sodium dichloroaniline phenylacetate, diflunisal, etodolac, phenoxyhydric calcium atropine, flurbiprofen, ibuprofen, indoclorox, ketoprofen, meclofenamate sodium, mefenamic acid, nabumetone, naproxen sodium, oxyphenbutazone, phenylbutzonone, piroxicam, sulindac, tolmetin sodium.

Method for carrying out the invention

A. Polypeptide and antibody preparation

The following description relates primarily to the production of the polypeptides or antibodies described herein by culturing cells transformed or transfected with a vector comprising a nucleic acid encoding the same polypeptide or antibodyCells, and purifying the produced protein or antibody. Of course, it is contemplated that alternative methods known in the art may be used to prepare such polypeptides or antibodies. For example, such sequences, or portions thereof, can 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, CA (1969); merrifield, j.am.chem.soc.,85：2149-2154(1963)]. In vitro protein synthesis can be performed using manual techniques or by automated means. Automated synthesis can be achieved, for example, using an Applied Biosystems peptide synthesizer (Foster City, Calif.) using the manufacturer's instructions. The various portions of the proteins or antibodies described herein can be chemically synthesized separately, combined using chemical or enzymatic methods.

1. Isolation of DNA encoding the proteins described herein

DNA encoding a protein described herein can be obtained from a cDNA library prepared from tissues thought to possess the corresponding mRNA and express it at detectable levels. Thus, such human protein-encoding DNA may be conveniently obtained from a cDNA library prepared from human tissue, as described in the examples. Protein-encoding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (e.g., oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest. Standard procedures can be used, for example as described in Sambrook et al, molecular cloning: a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) screened cDNA or genomic libraries with selected probes. An alternative method for isolating the gene encoding the desired gene is the PCR method [ Sambrook et al, supra; dieffenbach et al, PCR Primer: a Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995) ].

The following examples describe techniques for screening cDNA libraries. Selecting as a probeShould be long enough, and well-defined enough to minimize false positives. The oligonucleotides are preferably labeled so as to be detectable upon hybridization to the DNA in the library to be screened. Methods of marking are well known in the art and include the use of pigments such as³²Radiolabelling such as P-labelled ATP, biotinylation or enzymatic labelling. Hybridization conditions, including moderately stringent and highly stringent hybridization conditions, are provided in Sambrook et al, supra.

The sequences identified in such library screening methods can be compared and aligned with other known sequences that are stored in and available from public databases, such as Genbank or other proprietary sequence databases. The identity (amino acid level or nucleotide level) of sequences that are in a defined region of a molecule, or that span the full-length sequence, can be determined using methods known in the art and as described herein.

Nucleic acids having protein coding sequences can be first obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein, and if necessary, using conventional primer extension procedures as described in Sambrook et al, supra, to detect precursors and processing intermediates of mRNA that may not be reverse transcribed into cDNA.

2. Selection and transformation of host cells

Host cells transfected or transformed with an expression vector or cloning vector containing the protein or antibody to be produced as described herein are cultured in conventional media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Culture conditions, such as medium, temperature, pH, etc., can be selected by the skilled artisan without undue experimentation. In general, the method can be described in Mammalian Cell biotechnolgy: principles, protocols and Practical techniques for maximizing the productivity of cell cultures were found in Practical Approach, m.butler, ed. (IRLPress, 1991) and Sambrook et al, supra.

Eukaryotic cellMethods for cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, e.g., CaCl₂、CaPO₄Methods of liposome-mediated and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment with calcium chloride, or electroporation as described in Sambrook et al, supra, is generally used for prokaryotes. Such as the group of Shaw et al, Gene,23: 315(1983) and WO89/05859, published 29.6.1989, infection with Agrobacterium tumefaciens was used to transform certain plant cells. For mammalian cells without a cell wall, Graham and van der Eb, Virology,52: 456-457 (1978). The general case of mammalian cell host system transfection has been described in U.S. Pat. No.4,399,216. Generally according to Van Solingen et al, j.bact,130: 946(1977) and Hsiao et al, Proc. Natl. Acad. Sci. (USA)76: 3829(1979) to yeast. However, other methods for introducing DNA into cells may be used, such as nuclear microinjection, electroporation, fusion with bacterial protoplasts of intact cells, or polycation methods such as Polybrene, polyornithine. Various techniques for transforming mammalian cells are described in Keown et al, Methods in enzymology,185: 527- & 537(1990) and Mansour et al, Nature,336：348-352(1988)。

suitable host cells for cloning or expressing the DNA herein in a vector include prokaryotes, yeast or higher eukaryote cells. Suitable prokaryotes include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, e.g., the family of enterobacteriaceae, e.g., escherichia coli. Various Escherichia coli strains are publicly available, for example Escherichia coli K12 strain MM294(ATCC 31,446); escherichia coli X1776(ATCC 31,537); escherichia coli strains W3110(ATCC 27,325) and K5772(ATCC 53,635). Other suitable prokaryotic host cells include the Enterobacteriaceae family, such as the genus Escherichia, for example, Escherichia coli, Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella(Klebsiella), Proteus (Proteus), Salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia), such as Serratia marcescens (Serratia marcescens), and Shigella (Shigella), and Bacillus, such as Bacillus subtilis and Bacillus licheniformis (B.licheniformis) (e.g., Bacillus 41P disclosed in DD 266,710, published 4.12.1989), Pseudomonas, such as Pseudomonas aeruginosa (P.aeruginosa), and Streptomyces. These examples are illustrative and not restrictive. Strain W3110 is a particularly preferred host or parent host because it is a versatile host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes. For example, strain W3110 may be modified to cause genetic mutations in genes encoding proteins endogenous to the host, examples of such hosts include Escherichia coli W3110 strain lA2, which has the complete genotype tonA; escherichia coli W3110 strain 9E4, which has the complete genotype tonA ptr 3; escherichia coli W3110 strain 27C7(ATCC 55,244) having the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kan^r(ii) a Escherichia coli W3110 strain 37D6 with the complete genotype tonAPr 3 phoA E15(argF-lac)169degP ompTs 7 ilvG kan^r(ii) a Escherichia coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and the Escherichia coli strain having a particular periplasmic protease disclosed in U.S. Pat. No.4,946,783 published on 7/8 in 1990. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for cloning or expressing vectors encoding FGF-19. Saccharomyces cerevisiae, or commonly used baker's yeast, is most commonly used among lower eukaryotic host microorganisms. Other eukaryotic microorganisms include: schizosaccharomyces pombe (Schizosaccharomyces pombe) (Beach and Nurse, Nature, 290: 140[1981 ]; EP 139383 published on 2.5.1985); kluyveromyces (Kluyveromyces) hosts (U.S. Pat. No. 4943529; Fleer et al, Bio/Technology, 9: 968-975(1991)), such as Kluyveromyces lactis (L.lactis) (MW98-8C, CBS683, CBS 4574; Louvocort et al, J.Bacteriol., 154 (2): 737 [1983]), Kluyveromyces fragilis (K.fragilis) (ATCC 12424), Kluyveromyces bulgaricus (K.bulbgaricus) (ATCC 16045), Kluyveromyces wilfordii (K.wickearrell) (ATCC24178), K.wallerti (ATCC 56500), Kluyveromyces drosophilus (K.sorbiarium) (ATCC 36906; Van Berg et al, Biotechnology, 8: Kluyveromyces (1990), Kluyveromyces thermosyphylla et al (K.thermosyphylla); yarrowia (EP 402226); pichia pastoris (EP 183070; Sreekrishna et al, J.basic Microbiol., 28: 265-278[1988 ]); candida species; trichoderma reesia (EP 244234); neurospora crassa (Case et al, Proc. Natl. Acad. Sci. USA, 76: 5259-5263[1979 ]); schwanniomyces such as Schwanniomyces occidentalis (EP 394538 published at 31.10.1990), and the like; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91/00357 published on 10.1.1991) and Aspergillus hosts such as Aspergillus nidulans (Ballance et al, biochem. Biophys. Res. Commun., 112: 284289[1983 ]; Tilburn et al, Gene, 26: 205-. Methylotropic (Methylotropic) yeasts are suitable herein, including but not limited to those selected from the following genera capable of growing in methanol: hansenula (Hansenula), Candida (Candida), Kloeckera (Kloeckera), Pichia (Pichia), Saccharomyces, Torulopsis, and Rhodotorula. A list of illustrative specific Saccharomyces species of such yeasts is found in C.Anthony, The biochemistry of genetics, 269 (1982).

Suitable host cells for expressing the glycosylated forms of the polypeptides and antibodies described herein are derived from multicellular organisms. Examples of invertebrate cells include insect cells (e.g., Drosophila S2 and Spodoptera Sf9) and plant cells. Useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells and COS cells. More specific examples include monkey kidney CV1 cell line transformed with SV40(COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 cells or subclones cultured into 293 cells grown in suspension culture, Graham et al, J.Gen Virol.36: 59 (1977)); chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); murine Sertoli cells (TM4, Mather, biol. reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and murine mammary carcinoma cells (MMT 060562, ATCC CCL 51). The selection of suitable host cells is common knowledge in the art.

3. Selection and use of replication-competent vectors

Nucleic acids (e.g., cDNA or genomic DNA) encoding the polypeptides or antibodies described herein can be inserted into replicable vectors for cloning (DNA amplification) or expression. Various vectors are publicly available. The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector using a variety of methods. Typically, the DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to: one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable vectors comprising one or more of these components are constructed using standard ligation techniques well known to those skilled in the art.

The recombinant product of the polypeptide or antibody can be prepared not only directly recombinantly, but also as a fusion polypeptide with a heterologous polypeptide, which is a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or may be part of the DNA encoding the polypeptide or antibody that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, 1pp, or a heat-stable enterotoxin II leader. For yeast secretion, there may be mentioned, for example, the yeast invertase leader, the alpha factor leader (including the alpha factor leaders of Saccharomyces and Kluyveromyces, the latter being described in U.S. Pat. No. 5010182), or the acid phosphatase leader, the Candida albicans glucoamylase leader (EP 362179 published 4/4 in 1990), or the signal sequence described in WO90/13646 published 11/15 in 1990. In mammalian cell expression, mammalian signal sequences can be used to directly secrete proteins, such as signal sequences derived from secreted polypeptides of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are well known in a variety of bacteria, yeasts and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin of replication is suitable for yeast, and the various viral origins of replication (SV40, polyoma, adenovirus, VSV or BPV) are suitable for cloning vectors in mammalian cells.

Expression and cloning vectors typically contain a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) provide resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, etc., (b) complement auxotrophic deficiencies, or (c) provide key nutrients not available from complex media, such as the gene encoding the Bacillus D-alanine racemase.

Examples of suitable selectable markers for mammalian cells are those that allow identification of cells, such as DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is one according to Urlaub et al, pric. 4216(1980), a DHFR activity deficient Chinese Hamster Ovary (CHO) cell line prepared and propagated by the method described in. Suitable selection genes for yeast use are the trp1 gene present in the yeast plasmid YRp7 [ Stinchcomb et al, Nature, 282: 39 (1979); kingsman et al, Gene, 7: 141 (1979); tschermer et al, Gene, 10: 157(1980)]. the trp1 gene provides a selectable marker for yeast mutants that cannot grow in tryptophan (e.g., ATCC44076 or PEP4-1) [ Jones, Genetics, 85: 12(1977)].

Expression and cloning vectors typically contain a promoter operably linked to the DNA sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems [ Chang et al, Nature, 275: 615 (1978); goeddel et al, Nature, 281: 544(1979), alkaline phosphatase, the tryptophan (trp) promoter system [ Goeddel, Nucleic acids res, 8: 4057 (1980); EP 36776], and hybrid promoters such as the tac promoter [ deBoer et al, proc.natl.acad.sci.usa, 80: 21-25(1983)]. Promoters suitable for use in bacterial systems also contain Shine-Dalgarno (S.D.) sequences operably linked to the DNA sequences.

Examples of suitable promoter sequences for use in a yeast host include 3-phosphoglycerate kinase [ Hitzeman et al, j.biol.chem., 255: 2073(1980) or other glycolytic enzymes [ Hess et al, j.adv.enzymereg, 7: 149 (1968); holl and biochemistry, 17: 4900(1978), and 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, i.e., those inducible promoters which have the advantage of transcription controlled by growth conditions, are the promoter regions of the genes ethanol 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 systems are further described in EP 73657.

In mammalian host cells, transcription of the vector can be regulated by promoters from viral genomes such as polyoma virus (polyoma virus), fowlpox virus (fowlpox virus) (UK 2211504 published on 5.7.1989), adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis b virus, and monkey virus 40(SV40), or from heterologous mammals such as actin promoter or immunoglobulin promoter, and from heat shock promoters, provided that these promoters are compatible with the host cell system.

Transcription of nucleic acids encoding the polypeptides or antibodies herein by higher eukaryotes may be increased by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase transcription, typically about 10-300 bp. Many enhancer sequences for mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are known. However, one typically uses an enhancer of a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of its replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of its replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector 5 ' or 3 ' to the coding sequence, but is preferably located 5 ' to the promoter.

Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) also include sequences necessary for transcription termination and stabilization of mRNA structure. These sequences are usually derived from the 5 '(and occasionally 3') untranslated region of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide fragments that are transcribed as polyadenylated fragments in the untranslated region of the mRNA encoding the polypeptides or antibodies described herein.

Other suitable methods, vectors and host cells suitable for synthesizing the polypeptides or antibodies described herein in recombinant vertebrate cell culture are described in Gething et al, Nature, 293: 620-625 (1981); mantei et al, Nature, 281: 40-46 (1979); EP 117060 and EP 117058.

4. Detecting gene amplification/expression

Amplification and/or expression of the gene can be determined directly in the sample, e.g., by conventional techniques such as Southern blotting, Northern blotting to determine the amount of mRNA transcript using appropriate labeled probes according to the sequences provided herein [ Thomas, proc.natl.acad.sci.usa, 77: 5201-5205(1980) ], dot blot (DNA analysis) or in situ hybridization, it is possible to determine gene amplification and/or expression directly in the sample. Alternatively, antibodies capable of recognizing specific duplexes including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes are used. The antibodies are sequentially labeled and an assay is performed as to which site on the surface the duplex binds, such that antibodies bound to the duplex are detected based on the formation of the duplex on the surface.

Alternatively, for direct quantification of expressed gene products, gene expression is measured immunologically, such as immunohistochemical staining of cells or tissue sections and cell culture or humoral analysis. The antibody suitable for immunohistochemical staining and/or sample fluid analysis may be a monoclonal antibody or a polyclonal antibody, and may be prepared in any mammal. Conveniently, antibodies may be prepared against the polypeptides described herein, or against peptides synthesized from the DNA sequences provided herein, or against foreign sequences fused to DNA encoding such polypeptides and antibodies, and foreign sequences fused to DNA encoding specific antibody epitopes.

5. Polypeptide purification

The polypeptide may be recovered from the culture medium or from a host cell lysate. If the polypeptide is bound to the membrane, it is released from the cell membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage. Cells used to express the polypeptides or antibodies described herein are disrupted using various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or a lytic agent.

It may be desirable to purify the polypeptides or antibodies described herein from recombinant cellular proteins or other polypeptides. The following procedure is an illustrative suitable purification step: fractionating on an ion-exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or a cation-exchange resin such as DEAE; focusing chromatography; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex (Sephadex) G-75; passing through a protein a sepharose column to remove contaminants such as IgG; and a metal chelator column that binds epitope-tagged form of FGF-19. Various Methods of protein purification may be used, which are well known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); scopes, Protein Purification: principes and Practice, Springer-Verlag, New York (1982). The choice of purification step depends, for example, on the nature of the production process and the particular polypeptide or antibody produced.

B. Antibody preparation

In certain embodiments of the invention, the protein of choice is an antibody. The following are techniques for producing antibodies, including polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

(1) A polyclonal antibody.

Polyclonal antibodies are typically elicited in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl succinimido ester (bound via a cysteine residue), N-hydroxysuccinimide (bound via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR, where R and R¹Independently a lower alkyl group, is useful for binding the antigen of interest to a protein that is immunogenic in the species being immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's incomplete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic diphtheria ester (dicynomomycolate)). The selection of an immunization regimen can be made by one of skill in the art without undue experimentation.

Immunization of animals with the antigen, immunogenic conjugate, or derivative is achieved by injecting the solution intradermally at multiple locations, for example 100 μ g or 5 μ g of the protein or conjugate (for rabbits or mice, respectively) in combination with 3 volumes of complete Freund's adjuvant. One month later, 1/5 to 1/10 initial amounts of peptide or conjugate were boosted in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, animals were bled and sera were used for analysis of antibody titers. Animals were boosted until titers reached peak levels. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, aggregating agents such as alum (alum) are suitable for enhancing immune responses.

(2) A monoclonal antibody.

A monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody comprising the population is identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of different antibodies.

For example, monoclonal antibodies can be produced by a method described by Kohler et al, Nature,256: 495(1975) or may be prepared by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, e.g., a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be sensitized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(academic Press, 1986).

The immunizing agent generally includes an antigenic protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, and spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells [ Goding, monoclonal antibodies: principles and Practice, Academic Press, (1986) pages 59-103 ].

Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Usually rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), and the culture medium for hybridomas typically includes hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances inhibit the growth of HGPRT-deficient cells.

Preferred immortal myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Among these, preferred are murine myeloma Cell 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 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American type culture Collection, Manassus, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines have also 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)).

The culture medium in which the hybridoma cells are grown is analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The culture medium in which the hybridoma cells are cultured can be analyzed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and analytical methods are known in the art. For example, the compound can be produced by Munson et al, anal. biochem., 107: 220(1980) to determine binding affinity.

After identification of hybridoma cells producing antibodies of the desired specificity, affinity and/or activity, the clones are subcloned by restriction dilution procedures and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo, such as in ascites tumors of mammals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, the peritoneal fluid or the serum by conventional immunoglobulin purification steps, such as protein a sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, for example, in U.S. Pat. No.4,816,567 and the methods described above. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the murine antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then used to transfect host cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, or non-immunoglobulin producing myeloma cells, to facilitate isolation of the DNA from the host cellsMonoclonal antibodies are synthesized in recombinant host cells. Reviews on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al, curr. opinion in immunol,5: 256-charge 262(1993) and Pluckthun, Immunol.Revs.130：151-188(1992)。

In further embodiments, the methods may be developed from methods utilizing McCafferty et al, Nature,348: 552-554(1990) by the techniques described therein. Clackson et al, Nature,352: 624-,222: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. The subsequent publication describes the transformation of a single strand by strand shuffling (Marks et al, Biol/Technology,10: 779-783(1992)) and combinatorial infection and in vivo recombination as strategies for constructing large phage libraries (Waterhouse et al, nucleic acids Res.,21: 2265-2266(1993)) to produce high affinity (nM range) human antibodies. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

For example, homologous murine sequences can also be substituted by the coding sequences for human heavy and light chain constant regions (U.S. Pat. No.4,816,567; Morrison, et al, Proc. Natl Acad. Sci. USA,81: 6851(1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the non-immunoglobulin polypeptide coding sequence. Typically such non-immunoglobulin polypeptides replace the constant regions of an antibody, or may replace the variable regions of one antigen-binding site of an antibody, to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

The monoclonal antibodies described herein may be monovalent, methods of making them being well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains. Typically the heavy chain is truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residue may be replaced by another amino acid residue, or deleted, to prevent cross-linking. In vitro methods are also suitable for the production of monovalent antibodies. Digestion of the antibody to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

Chimeric or hybrid antibodies can also be prepared in vitro using known synthetic protein chemistry methods, including those that utilize cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable agents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutylimidate (methyl-4-mercaptobutyliminate).

(3) A humanized antibody.

The antibody of the present invention may further include a humanized antibody or a human antibody. Non-human (e.g., murine) humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab ', F (ab')₂Or other antigen-binding subsequences of antibodies) that contain minimal non-human immunoglobulin sequences. Humanized antibodies include human immunoglobulins (recipient antibody) in which recipient Complementarity Determining Region (CDR) residues are replaced by CDR residues of a non-human species antibody (donor antibody) such as mouse, rat, rabbit having the desired specificity, affinity, and performance. In some examples, Fv framework region residues of the human immunoglobulin are substituted for corresponding non-human residues. Humanized antibodies may also comprise residues that are not present in either the recipient antibody or the donor CDR or framework sequences. Typically, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions in which all or substantially all of the CDRs correspond to a corresponding portion of a non-human immunoglobulin and all or substantially all of the FR sequences are human immunoglobulin consensus sequences. The humanized antibody also optionally includes an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. 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)。

Methods for humanizing non-human antibodies are known to those skilled in the art. Generally, humanized antibodies have one or more amino acid residues introduced into them that are derived from a non-human source. These non-human amino acid residues are often referred to as "introduced" residues, which are typically derived from an "introduced" variable region. Humanization was carried out essentially as described by 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 one or more CDR sequences for the corresponding sequences of the human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. patent 4816567) in which a substantial portion of the entire human variable region is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable regions, including light and heavy chain variable regions, in making humanized antibodies is very important to reduce antigenicity. The variable region sequences of rodent antibodies are screened against a complete library of known human variable region sequences according to the so-called "best-fit" method. The human sequence closest to the rodent sequence is then used as the human Framework (FR) of the humanized antibody. Sims et al, J.151: 2296 (1993); chothia et al, J.mol.biol.,196: 901(1987). Another approach uses a specific framework derived from the consensus sequence of human antibodies of a specific subgroup of light or heavy chains. The same framework can 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).。

more importantly, the antibodies are humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are well known to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the role residues may play in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect 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 to achieve desired antibody properties, such as increased affinity for the antigen of interest. Generally, CDR residues directly and very substantially (most substentiaily) affect antigen binding.

Various forms of humanized antibodies are contemplated by the present invention. For example, the humanized antibody may be an antibody fragment, such as a Fab, that selectively binds to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

(4) Human antibodies

As an alternative to humanization, human antibodies may be produced. For example, transgenic animals (e.g., mice) can be prepared that can be immunized to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been indicated that in chimeric and germ line (germ-line) mutant mice, the antibody heavy chain joining region (J)_H) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array to this germline mutant mouse will result in the production of human antibodies against antigen challenge. See, for example, Jakobovits et al, proceedings of the national academy of sciences USA, 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258 (1993); (ii) a U.S. Pat. No.5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce human antibodies or antibody fragments in vitro from repertoires of immunoglobulin variable (V) region genes from non-immunized donors. McCafferty et al, Nature348: 552 and 553 (1990); hoogenboom and Winter, j.227: 381(1991). According to this technique, antibody V region genes are cloned in-frame to the major and minor coat protein genes of filamentous phages, such as M13 or fd, presented as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection for functional properties of the antibody also results 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 various formats, for example, as described in Johnson, Kevin s. and Chiswell, David j., curr.3: 564-571(1993) is reviewed. Several sources of V gene fragments can be used for phage display. Clackson et al, Nature352: 624-628(1991) an array of a variety of anti-oxazolone antibodies (array) was isolated from a small random combinatorial library of V genes derived from the spleen of immunized mice. Marks et al, J.mol.biol.222: 581-597(1991), or Griffith et al, EMBO J.12: 725-734(1993) can construct all the components of the V gene from non-immunized human donors and isolate substantially antibodies against a variety of antigens, including autoantibodies. See, U.S. Pat. Nos. 5,565,332 and 5,573,905.

The techniques of Cole et al and Boerner et al are also available for the preparation of human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77(1985) and Boerner et al, J.Immunol.147(1): 86-95(1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which endogenous immunoglobulin genes have been partially or completely inactivated. At challenge, human antibody products were observed, which in all respects were similar to those seen in humans, including gene rearrangement, assembly, and antibody repertoire (antibody repertoire). This method is described, for example, in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016, and published in the following scienceAn object: marks et al, Bio/Technology10: 779 783 (1992); lonberg et al Nature368：856-859(1994)；Morrison，Nature 368: 812-13(1994), Fishwild et al, Nature Biotechnology14：845-51(1996)，Neuberger，Nature Biotechnology14: 826(1996) and Lonberg and Huszar, Intern.Rev.Immunol.13: 65-93 (1995).

Finally, human antibodies can also be produced in vitro by activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

(5) Antibody fragments

In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. Smaller fragment sizes allow for rapid clearance, which may result in increased exposure to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, j. biochem biophysis.24: 107-117 (1992); and Brennan et al, Science229: 81(1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments are all capable of expression in and secretion from Escherichia coli, thus allowing for easy production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries as described above. Alternatively, Fab '-SH fragments can be recovered directly from Escherichia coli and chemically ligated to form F (ab')₂Fragments (Carter et al, Bio/Technology)10: 163-167(1992)). According to another method, F (ab')₂And (3) fragment. Fab and F (ab') 2 with increased in vivo half-life are described in U.S. Pat. No.5,869,046. In other embodiments, the alternative antibody 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.Such linear antibody fragments may be monospecific or bispecific.

(6) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the invention may also be used in ADEPT by combining the antibody with a prodrug-activating enzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) into an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme that acts on a prodrug in a manner to convert it to its more active, cytotoxic form.

Enzymes useful in the methods of the invention include, but are not limited to, glycosidases, glucose oxidase, human lysozyme, human glucuronidase, 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 to the anticancer drug 5-fluorouracil; proteases, such as Serratia proteases, thermolysins, subtilisins, carboxypeptidases (e.g., carboxypeptidase G2 and carboxypeptidase A), and cathepsins (e.g., cathepsins B and L), are useful for converting peptide-containing precursor drugs to free drugs; d-alanylcarboxypeptidases useful for converting prodrugs comprising D-amino acid substituents; carbohydrate-cleaving enzymes, such as β -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; a beta-lactamase useful for converting a beta-lactam derived drug into a free drug; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, are useful for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups, respectively, at the amine nitrogen to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to conjugate the precursors of the inventionThe drug is converted to a free active drug (see, e.g., Massey, Nature328: 457-458(1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of abzymes into tumor cell populations.

The above enzymes can be covalently linked to the polypeptides or antibodies described herein by techniques well known in the art, for example, using the heterobifunctional cross-linking agents described above. Alternatively, fusion proteins comprising at least the antigen-binding region of an antibody of the invention linked to at least one functionally active portion of an enzyme of the invention can be prepared using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al, Nature312：604-608(1984))。

7) Bispecific and multispecific antibodies

Bispecific antibodies (BsAb) are those antibodies that have binding specificities for at least two different epitopes, including those epitopes on the same or another protein. Alternatively, one arm may be equipped to bind to the target antigen and the other arm may be combined with an arm that binds to a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3) or Fc receptors for IgG (Fc γ R), such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16), to focus and localize cellular defense mechanisms to cells expressing the target antigen. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F (ab') 2 bispecific antibodies).

Bispecific antibodies can also be used to target cytotoxic agents to cells expressing the antigen of interest. Such antibodies possess one arm that binds to the desired antigen and another arm that binds to the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkoloid, ricin a chain, methotrexate, or radioisotope hapten). Examples of known bispecific antibodies include anti-ErbB 2/anti-fcgii (WO 96/16673), anti-ErbB 2/anti-FcgRI (U.S. patent 5,837,234), anti-ErbB 2/anti-CD 3 (U.S. patent 5,821,337).

Manufacture of bispecific antibodiesMethods for sex antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities. Millstein et al, Nature,305: 537-539(1983). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, usually by an affinity chromatography step, is rather cumbersome and the product yield is low. In WO 93/08829 and in Traunecker et al, EMBO J,10: 3655-3659(1991) disclose similar procedures.

According to different methods, an antibody variable region with the desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. The fusion is preferably to an immunoglobulin heavy chain constant region, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred that a first heavy chain constant region (CH1) having a site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion, and if desired the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. While unequal ratios of the three polypeptide chains provide optimal yields for construction, in embodiments this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in equal ratios resulting in high yields, or when said ratios have no particular significance, it is possible to insert two or all three polypeptide chain encoding sequences into one expression vector.

In a preferred embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. It was found that this asymmetric structure facilitates the isolation of the desired bispecific from unwanted immunoglobulin chain bindersThe presence of an immunoglobulin light chain in only half of the bispecific molecule provides an easy separation route. This process is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al, Methods in Enzymology121：210(1986)。

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between the pair of antibody molecules can be designed to maximize the percentage of heterodimers recovered from recombinant cell cultures. Preferred contacting surfaces comprise at least a portion of the CH3 region of the antibody constant region. In this method, one or more small amino acid side chains from the contact face of the first antibody molecule are replaced by large side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with small amino acid side chains (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the large side chains is created on the contact surface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesired end products, such as homodimers.

Methods for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science229: 81(1985) describes a procedure in which intact antibodies are proteolytically cleaved to yield F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dimercaptoxide complexing agent sodium arsenite to stabilize adjacent dimercaptotes and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Trinitrobenzene (TNB) derivatives. One of the Fab '-TNB derivatives is then converted back into the Fab' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Fab' fragments can be recovered directly from E.coli and chemically coupled to form bispecific antibodies. Shalaby et al, j.175：217-225(1992) Fully humanized bispecific antibodies F (ab')₂And (3) producing molecules. Each Fab' fragment was separately secreted from E.coli and directly chemically coupled in vitro to form bispecific antibodies. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells and was able to trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for the production and isolation of bivalent antibody fragments directly from recombinant cell cultures have also been described. For example, a bivalent heterodimer has been generated using a leucine zipper structure. Kostelny et al, J.Immunol.,148(5): 1547-1553(1992). The leucine zipper structural peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-6448(1993) describe the "diabody" technology to provide an alternative mechanism for the production of bispecific/bivalent antibody fragments. The fragment comprises a variable region (V) linked to a light chain via a linker_L) Heavy chain variable region (V) of_H) The linkers are so short that pairing between two regions of the same strand is not allowed. Thus, V of a segment_HAnd V_LThe region has to be complementary V to another fragment_LAnd V_HThe regions pair, thereby forming two antigen binding sites. Another strategy for making bispecific/bivalent antibody fragments using single chain fv (sfv) dimers has been reported. See Gruber et al, j.immunol.,152：5368(1994)。

antibodies with more than two valencies are also within the scope of the invention. For example, trispecific antibodies may be prepared. Tutt et al, j.147：60(1991)。

An exemplary bispecific antibody can bind to two different epitopes on a given molecule. Alternatively, an anti-protein arm may be combined with an arm that binds a trigger molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor of IgG (fcyr), such as fcyri (CD64), fcyrii (CD32), and fcyriii (CD16), to focus the defense mechanism of the cell on cells expressing a particular protein. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing a particular protein. Such antibodies possess a protein-binding arm and an arm that binds to a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to a protein of interest and further binds to Tissue Factor (TF).

6) Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies. For example, one antibody in a heteroconjugate can be conjugated to avidin and the other to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells, U.S. Pat. No.4,676,980, and for treating HIV infection. WO 91/00360, WO92/200373 and EP 0308936. It is contemplated that the antibodies may also be prepared in vitro using known synthetic protein chemistry methods, including those using cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidates, and those disclosed, for example, in U.S. Pat. No.4,676,980. Heteroconjugate antibodies can be made using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

(7) Effector function modification

It may be desirable to modify the antibodies of the invention according to effector function in order to enhance the effectiveness of the antibodies in treating cancer. For example, cysteine residues may be introduced into the Fc region, allowing interchain disulfide bonds to form in this region. Homodimerization antibody thus producedHave increased internalization capacity 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 shop, J.148: 2918-2922(1992). Also, for example, Wolff et al, Cancer Research may be utilized53: 2560-2565(1993) to prepare homodimeric antibodies with enhanced anti-tumor activity. Alternatively, the antibody can be designed to have a dual Fc region, such that the antibody has enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design3：219-230(1989)。

(8) Immunoconjugates

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., a bacterial, fungal, plant or animal enzymatically active toxin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful for generating such immunoconjugates include BCNU, streptozoicin, vincristine, vinblastine, doxorubicin and 5-fluorouracil.

Enzymatically active toxins and fragments thereof that may be used include: diphtheria toxin a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, alpha-sarcin, aleurites fordii protein, dianthin protein, phytolacca Americana protein (PAPI, PAPII, PAP-S), momordica charantia (momordia) inhibitor, curcin, crotin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, mitomycin (mitogellin), restrictocin, phenomycin, enomycin, and trichothecenes (tricothecenes).

Conjugates of the antibody and cytotoxic agent may be attached by a variety of bifunctional protein coupling agents such as: n-succinimidyl-3-, (2-pyridyldimercapto) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (it), bifunctional derivatives of imidoesters (e.g., dimethyl imidoadipate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azides (e.g., bis (p-azidobenzoyl) hexanediamine), bis-diazo derivatives (e.g., bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (e.g., tolylene 2, 6-diisocyanate), and bis-active fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin may be identified as vietta et al, science 238: 1098 (1987). C¹⁴Labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetate (MX-DTPA) is one of the coupling agents for coupling radionucleotides to antibodies. See WO 94/11026. Such a linker may be a "breakable linker" which facilitates release of the cytotoxic drug within the cell. For example, acid labile linkers, peptidase-sensitive linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, cancer Res. 52: 127- "131 (1992)).

In addition, small molecule toxins such as calicheamicin (calicheamicin), maytansine (maytansine) (U.S. Pat. No.5,208,020), trichothene, and CC1065 are also contemplated as conjugative toxins for use in the formulations of the present invention. In one embodiment, a full-length antibody or antigen-binding fragment thereof can bind to one or more maytansinoid (maytansinoid) molecules (e.g., about 1-10 maytansinoid molecules per antibody molecule). Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansinoids, including maytansine, maytansinoids and derivatives and analogs thereof, isolated from natural sources or prepared synthetically have been described, see U.S. Pat. No.5,208,020 and references cited therein (see column 2, line 53 to column 3, line 10) and U.S. Pat. Nos. 3,896,111 and 4,151,042. Methods of preparing antibody-maytansinoid conjugates are also described in U.S. Pat. No.5,208,020. In a preferred embodiment, the maytansinoid is produced by disulfideThe conjugate or other sulfur-containing linker group is attached to the antibody. For example, maytansine May be converted to May-SS-Me, which May be reduced to May-SH3 and reacted with a modified antibody to produce a maytansinoid-antibody immunoconjugate. Chari et al, Cancer Res.52: 127-131(1992). The antibody may be modified by known methods, and the antibody containing a free or protected thiol group is then reacted with a maytansinoid containing a disulfide to produce the conjugate. The cytotoxicity of the antibody-maytansinoid conjugates can be determined in vitro or in vivo by known methods and can be measured using IC₅₀And (4) determining.

Calicheamicin (calicheamicin) is another immunoconjugate of interest. The calicheamicin family of antibiotics produce double-stranded DNA breaks at sub-picomolar concentrations. Structural analogs of calicheamicin that may be used include, but are not limited to, gamma₁ ¹、α₂ ¹、α₃ ¹N-acetyl-gamma₁ ¹PSAG and θ¹ ₁(Hinman et al, Cancer Res.53: 3336 Astro 3342(1993) and Lode et al, Cancer Res.58: 2925-2928(1998)). Other antineoplastic agents to which antibodies may be conjugated include QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents by antibody-mediated internalization greatly enhances their cytotoxic effects.

Immunoconjugates formed between an antibody and a compound having nucleolytic activity (e.g., ribonuclease or DNA endonuclease, e.g., deoxyribonuclease, DNase)) are also contemplated.

Antibodies may also be conjugated to highly radioactive atoms. Various radionuclides for the production of radioconjugated antibodies are available. Examples include At²¹¹、Bi²¹²、I¹³¹、In¹³¹、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、P³²And Pb²¹²And radioactive isotopes of Lu. When the conjugate is usedIn diagnosis, it may contain radioactive atoms, such as Tc, for scintigraphic studies⁹⁹Or I¹²³Or spin labels for nuclear magnetic resonance (nmr) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Radiolabels or other labels may be incorporated into the conjugates using known methods. For example, the peptides may be biosynthesized or chemically synthesized by chemical amino acid synthesis using suitable amino acid precursors comprising, for example, fluorine-19 or hydrogen at the appropriate position. Tc⁹⁹Or I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹The label may be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via lysine residues.The method can be used to incorporate iodine-123, Fraker et al, Biohem. Biophys. Res. Commun.80: 49-57(1978). Other methods of binding radionuclides are described in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

Alternatively, fusion proteins comprising an antibody and a cytotoxic agent can be made by recombinant techniques or peptide synthesis. The length of the DNA may comprise respective regions encoding the two parts of the conjugate, which regions may be adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In another embodiment, the antibody can be conjugated to a "receptor" (e.g., streptavidin) for use in the pre-targeting of tumors, wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from circulation with a clearing agent, followed by administration of a "ligand" (e.g., avidin) that binds to a cytotoxic agent (e.g., a radionucleotide).

(9) Immunoliposomes

The antibodies disclosed herein can also be formulated as immunoliposomes. "liposomes" are small vesicles composed of various types of lipids, phospholipids and/or surface active substances and are useful for the delivery of drugs to mammals. The components of liposomes are typically arranged in a bilayer formation, similar to the lipid profile of biological membranes.

Antibody-containing liposomes are prepared by methods known in the art, such as, for example, Epstein et al, Proc.Natl.Acad.Sci.USA,82: 3688 (1985); hwang et al, Proc.Natl Acad.Sci.USA,77: 4030 (1980); and the methods described in U.S. patent nos. 4,485,045 and 4,544,545. Liposomes having enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter material of defined pore size to produce liposomes of a desired diameter. Such as Martin et al, J.biol.chem.,257: 286- "288 (1982) by disulfide exchange to bind the Fab' fragments of the antibodies of the invention to liposomes. Optionally, a chemotherapeutic agent (e.g., doxorubicin) is contained within the liposome. See Gabizon et al, J.national Cancer Inst.81(19)：1484(1989)。

(10) Other antibody modifications

Other modifications to the antibodies are contemplated herein. For example, the antibody can be linked to various non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. The antibody may also be encapsulated in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsions prepared, for example, by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly- (methylmethacylate) microcapsules, respectively). In Remington: this technique and suitable formulations are disclosed in the Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia college of Pharmacy and Science 2000).

C. Freeze-dried preparation

The formulations described herein may also be prepared as reconstituted lyophilized formulations. The proteins or antibodies described herein are lyophilized and then reconstituted to produce the reduced viscosity stable liquid formulations of the present invention. In this particular embodiment, the "pre-lyophilized formulation" is produced after the protein of interest is prepared as described above. The amount of protein present in the pre-lyophilized formulation is determined in view of the desired dosage, the mode of administration, and the like. For example, the starting concentration of intact antibody may be from about 2mg/ml to about 50mg/ml, preferably from about 5mg/ml to about 40mg/ml and most preferably about 20-30 mg/ml.

(1) Preparation of lyophilized preparation

The protein to be formulated is generally present in solution. For example, in the formulations of the invention having increased ionic strength and decreased viscosity, the protein may be present in a pH buffered solution at a pH of about 4 to about 8, preferably about 5 to about 7. Depending on, for example, the buffer and the desired formulation tension (e.g., the tension of the reconstituted formulation), the buffer concentration may be from about 1mM to about 20mM, alternatively from about 3mM to about 15 mM. Exemplary buffers and/or salts are those that are pharmaceutically acceptable and can be generated from suitable acids, bases, and salts thereof, such as those defined by "pharmaceutically acceptable".

In one embodiment, a lyoprotectant is added to the pre-lyophilized formulation. The amount of lyoprotectant dissolved in the pre-lyophilized formulation is generally such that upon reconstitution the resulting formulation is isotonic. However, hypertonic reconstituted formulations may also be suitable. Furthermore, the amount of lyoprotectant cannot be so low that unacceptable amounts of protein degradation/aggregation occur upon lyophilization. However, exemplary lyoprotectant concentrations in the pre-lyophilized formulation are from about 10mM to about 400mM, alternatively from about 30mM to about 300mM, alternatively from about 50mM to about 100 mM. Exemplary lyoprotectants include sugars and sugar alcohols, such as sucrose, mannose, trehalose, glucose, sorbitol, mannitol. However, certain solubility protectors may also increase the viscosity of the formulation under certain circumstances. Likewise, care should be taken to select a particular lyoprotectant that minimizes and neutralizes these effects. Under the definition of "lyoprotectant", additional lyoprotectants are described above, also referred to herein as "pharmaceutically acceptable sugars".

The ratio of protein to lyoprotectant may vary for each particular protein or antibody and lyoprotectant combination. For the case of antibodies as candidate proteins and sugars (e.g., sucrose or trehalose) as lyoprotectants to produce an isotonic reconstituted formulation of high protein concentration, the molar ratio of lyoprotectant to antibody may be from about 100 to about 1500 moles lyoprotectant to 1 mole antibody, and preferably from about 200 to about 1000 moles lyoprotectant to 1 mole antibody, e.g., from about 200 to about 600 moles lyoprotectant to 1 mole antibody.

In a preferred embodiment, it is desirable to add a surfactant to the pre-lyophilized formulation (pre-lyophilised formulation). Alternatively, or in addition, a surfactant may be added to the lyophilized formulation and/or the reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20 or 80); polyoxamers (e.g., poloxamer 188); triton; sodium octyl glucoside; dodecyl-, tetradecyl-, linoleyl-, or stearyl-thiobetaine (sulfobetaine); dodecyl-, tetradecyl-, linolenyl-, or stearoyl-sarcosine; linoleyl-, tetradecyl-, or hexadecyl-betaine; lauramidopropyl (lauroamidopropyl) -, cocamidopropyl (cocamidopropyl) -, linoleamidopropyl (linoleamidopropyl) -, myristomidopropyl (myristomidopropyl) -, palmidopropyl (palmidopropyl) -, or isostearamidopropyl) -betaine (e.g. lauramidopropyl); myristamidopropyl-, palmAmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate (sodium methyl cocoyl-taurate) or disodium methyl oleyl taurate (sodium methyl oleyl-taurate); and MONAQUA^TMSeries (Mona Industries, inc., Paterson, new jersey), polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). The amount of surfactant added is such that it reduces the microparticle formation of the reconstituted protein and minimizes the microparticle formation after reconstitution. For example, the surfactant may be present in the pre-lyophilized formulation in an amount of about 0.001-0.5%, alternatively about 0.005-0.05%.

Mixtures of dissolution protectants (e.g., sucrose or trehalose) and bulking agents (e.g., mannitol or glycine) may be used in the preparation of the pre-lyophilized formulation. The bulking agent can allow for the creation of a homogeneous lyophilized cake without excess voids therein. Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's pharmaceutical Sciences 16th edition, Osol, a.ed. (1980), may be included in the pre-lyophilized formulation (and/or lyophilized and/or reconstituted) so long as they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: an additional buffering agent; a preservative; an auxiliary solvent; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counter ions, such as sodium.

The formulations herein may also comprise more than one protein, preferably those proteins having complementary activity that do not adversely affect another protein, as long as it is necessary for the particular condition to be treated. For example, it may be desirable to provide two or more antibodies that bind to a desired target (e.g., receptor or antigen) in a single formulation. Such proteins are suitably present in the composition in an amount effective for the intended target.

Formulations for in vivo administration must be sterile. This can be readily accomplished by filtration through a sterile filtration membrane, either before or after lyophilization and reconstitution. Alternatively, for example, sterilization of the complete mixture can be achieved by autoclaving the components other than the protein at about 120 ℃ for 30 minutes.

After the protein, optional lyoprotectant, and other optional components are mixed together, the formulation is lyophilized. For this purpose, a number of different freeze-dryers are available, for example Hull50^TM(Hull, USA) or GT20^TM(Leybold-Heraeus, Germany) freeze-dryer. Lyophilization is achieved by freezing the formulation, followed by sublimation of ice from the frozen contents at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or slump temperature (collapse temperature) of the formulation. Typically, the storage temperature (shelf temperature) for primary drying ranges from about-30 ℃ to 25 ℃ (provided the product remains frozen during primary drying), under appropriate pressure, typically ranging from about 50 to 250 mTorr. The formulation, size and type of container holding the sample (e.g., glass vial), and volume of liquid will primarily determine the time required for drying, which may range from a few hours to a few days (e.g., 40-60 hours). Optionally, a second drying may also be performed depending on the desired residual moisture level in the product. The temperature at which the second drying is carried out ranges from about 0 to 40 c, depending mainly on the type and size of the container and the type of protein used. For example, the storage temperature may be about 15-30 ℃ (e.g., about 20 ℃) throughout the water removal stage of lyophilization. The time and pressure required for the second drying is that which will produce a suitable lyophilized cake, depending, for example, on temperature and other parameters. The second drying time is determined by the desired residual moisture level in the product and generally takes at least about 5 hours (e.g., 10-15 hours). The pressure may be the same as used during the primary drying step. The lyophilization conditions may vary depending on the formulation and vial size.

2. Reconstitution of lyophilized formulations

Prior to administration to a patient, the lyophilized formulation is reconstituted with a pharmaceutically acceptable diluent such that the protein concentration in the reconstituted formulation is at least about 50mg/ml, e.g., from about 50mg/ml to about 400mg/ml, alternatively from about 80mg/ml to about 300mg/ml, alternatively from about 90mg/ml to about 150 mg/ml. Such high protein concentrations in reconstituted formulations are considered particularly useful when it is desired to deliver the reconstituted formulation subcutaneously. However, for other routes of administration, such as intravenous administration, lower protein concentrations in the reconstituted formulation may be desirable (e.g., about 5-50mg/ml, or about 10-40mg/ml of protein in the reconstituted formulation). In certain embodiments, the concentration of protein in the reconstituted formulation is significantly higher than the concentration in the pre-lyophilized formulation. For example, the concentration of protein in the reconstituted formulation may be about 2-40 times, alternatively 3-10 times, alternatively 3-6 times (e.g., at least three times or at least four times) the concentration of protein in the pre-lyophilized formulation.

Reconstitution generally occurs at about 25 ℃ to ensure complete hydration, although other temperatures may be used freely. The time required for reconstitution depends on, for example, the type of diluent, the number of excipients, and the protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. The diluent optionally comprises a preservative. Exemplary preservatives have been described above, aromatic alcohols such as benzyl alcohol or phenol alcohol being preferred preservatives. The amount of preservative used is determined by assessing the compatibility of the different preservative concentrations to the protein and preservative efficiency tests. For example, if the preservative is an aromatic alcohol (e.g., benzyl alcohol), the amount can be about 0.1-2.0% and preferably about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, the reconstituted formulation has less than 6000 particles per vial, and a particle size of 10 μm or more.

D. Liquid preparation

Therapeutic formulations for preservation are prepared by mixing the active ingredient with the desired purity, optionally with pharmaceutically acceptable carriers, excipients or stabilizers (Remington's pharmaceutical sciences 18th edition, Mack Publishing co., Easton, pa.18042[1990 ]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants, including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.

When the therapeutic agent is an antibody fragment, a minimal inhibitory fragment that specifically binds to the binding region of the protein of interest is preferred. For example, depending on the variable region sequence of the antibody, antibody fragments, or even peptide molecules, can be designed to retain the ability to bind to the target protein sequence. Such peptides may be chemically synthesized and/or produced by recombinant DNA techniques (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA)90：7889-7893[1993])。

Buffers are used to control the pH within a range that optimizes efficacy, particularly where stability is pH dependent. The preferred concentration of the buffer ranges from about 50mM to about 250 mM. Suitable buffers for use in the present invention include organic and inorganic acids, and their salts. For example citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may consist of histidine and a trimethylamine salt, such as Tris.

Preservatives are added to inhibit microbial growth, typically in the range of 0.2% to 1.0% (w/v). Suitable preservatives for use in the present invention include, for example, octadecyl dimethyl phenyl ammonium chloride; quaternary ammonium chloride hexahydrocarbons; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl carbinol or benzyl alcohol; hydrocarbyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol. .

A tonicity agent (sometimes referred to as a "stabilizer") is included to adjust or maintain the liquid tonicity of the composition. When large, charged biomolecules such as proteins and antibodies are used, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intra-and intermolecular interactions. The tonicity agent may be present in any amount between 0.1% and 25% by weight, preferably between 1 and 5% by weight, taking into account the relative amounts of the other ingredients. Tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Additional excipients include agents that can act as one or more of the following: (1) a filler, (2) a dissolution enhancer, (3) a stabilizer, and (4) an agent that inhibits denaturation or adhesion to a container wall. The stabilizer may range from 0.1 to 10,000 parts by weight of active protein or antibody. Typical stabilizers include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoionitose, myoionitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thiosulfate, thioglycerol, α -monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose), trisaccharides, such as raffinose, and polysaccharides such as dextrins or dextrans.

The inclusion of a non-ionic surfactant or detergent (also referred to as a "wetting agent") to aid in the dissolution of the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, also allows the formulation to be subjected to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant ranges from about 0.05mg/ml to about 1.0mg/ml, preferably from about 0.07mg/ml to about 0.2 mg/ml.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.),polyols，polyoxyethylene sorbitan monoethers (polyoxyethylenesorbitan monoethers) (Tween-20, Tween-80, etc.), lauromaprogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil (polyoxyethylenehydrogenated castor oil)10, 50 and 60, glyceryl monostearate, fatty acid sugar esters, methyl cellulose and carboxymethyl cellulose. Anionic detergents that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate (dioctyl sodium sulfosuccinate) and dioctyl sodium sulfosuccinate (dioctyl sodium sulfosuccinate). Cationic detergents include benzalkonium chloride (benzalkonium chloride) or benzethonium chloride (benzathinum chloride).

In order for the formulations to be used for in vivo administration, they must be sterile. The formulation may be sterilized by filtration through a sterile filtration membrane. The therapeutic compositions herein are generally placed into a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is according to known and recognized methods, for example by bolus administration (bolus) or by infusion over a prolonged period of time in a suitable manner, for example by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, by topical administration, by inhalation or by sustained or delayed release methods.

The formulations herein may also contain more than one active compound, preferably those having complementary activities that do not adversely affect each other, as long as they are necessary for the particular condition being treated. Alternatively, or in addition, the composition may include a cytotoxic agent, cytokine, or growth inhibitory agent. Such molecules are suitably present in the composition in an amount effective for the intended target.

The active ingredient may also be encapsulated in microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsions prepared, for example, by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly- (methylmethacylate) microcapsules, respectively). This technique is disclosed in Remington's pharmaceutical sciences 18th edition, supra.

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl ethanol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Recombinant protein microencapsulation for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2 and MN rpg 120. Med, Johnson et al, nat.2: 795-799 (1996); yasuda et al, biomed.27: 1221-1223 (1993); hora et al, Bio/Technology8：755-758(1990)；Cleland，″Design and Production of Single Immunization Vaccines Using PolylactidePolyglySlide Microsphere Systems, "in Vaccine Design: the Subunit and dAdjuvant Approach, Powell and Newman, eds., (Plenum Press: New York, 1995), pp.439-462; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. patent No.5,654,010.

Sustained release formulations of these proteins can be developed using polylactic-co-glycolic acid (PLGA) polymers because PLGA has biocompatibility and a wide range of biodegradable properties. Degradation products of PLGA, lactic acid and glycolic acid, are rapidly cleared from the human body. Furthermore, the degradability of this polymer can be adjusted from months to years, depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from active/polysaccharide Polymers", in Biodegradable Polymers as Drug delivery systems (Marcel Dekker; New York, 1990), M.Chasin and R.Langer (Eds.) pp.1-41.

Polymers such as ethylene-vinyl acetate and lactic-glycolic acid allow release of molecules for over 100 days, with some hydrogels releasing proteins in a shorter period of time. When the encapsulated antibodies are maintained in vivo for a long period of time, they may denature or aggregate as a result of exposure to a humid environment at 37 ℃, resulting in loss of biological activity and possibly a change in immunogenicity. Depending on the mechanism involved, a reasonable strategy can be devised for stability. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, utilizing appropriate additives, and developing specific polymer matrix compositions.

Liposomes or proteinaceous compositions can also be used to formulate the proteins or antibodies disclosed herein. See U.S. Pat. Nos. 4,925,673 and 5,013,556.

The stability of the proteins and antibodies described herein can be enhanced by the use of non-toxic "water-soluble multivalent metal salts". Examples include Ca²⁺、Mg²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Cu²⁺、Sn²⁺、Sn⁴⁺、Al²⁺And Al³⁺. Examples of anions that can form water-soluble salts with the above polyvalent metal cations include those formed from inorganic and/or organic acids. The water-soluble salt has a solubility in water (20 ℃) of at least about 20mg/ml, alternatively at least about 100mg/ml, alternatively at least about 200 mg/ml.

Suitable inorganic acids that may be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that may be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids under this definition may be defined as saturated or unsaturated C_2-9Carboxylic acids (e.g., aliphatic mono-, di-, and tricarboxylic acids). For example, exemplary monocarboxylic acids under this definition include saturated C_2-9Monocarboxylic acids, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capryonic acid, and unsaturated C_2-9Monocarboxylic acids, acrylic acid, preprolic methacrylic acid, crotonic acid and isocrotonic acid. Exemplary dicarboxylic acids include saturated C_2-9Dicarboxylic acids, malonic, succinic, glutaric, adipic and pimelic acids, and unsaturated C_2-9Dicarboxylic acids include maleic acid, fumaric acid, citraconic acid, and mesaconic acid. Exemplary tricarboxylic acids include saturated C_2-9Tricarboxylic acids, tricarballylic acid and 1, 2, 3-butanetricarboxylic acid. In addition, carboxylic acids of this definition also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, hydroxyglutaric acid, malic acid, tartaric acid, and citric acid. Aromatic acids under this definition include benzoic acid and salicylic acid.

Commonly used water-soluble multivalent metal salts that can be used to help stabilize the encapsulated polypeptides of the invention include, for example: (1) inorganic metal salts of halogens (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) metal salts of aliphatic carboxylic acids (e.g., calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) aromatic carboxylic acid metal salts, benzoates (e.g., zinc benzoate) and salicylates.

E. Medical and metallurgical method

For the prevention or treatment of a disease, the appropriate dosage of the active agent will depend upon the type of disease to be treated, as defined above, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's history and response to the agent, and the judgment of the attending physician. The medicament is suitably administered to the patient at one time or over a series of treatments.

The preferred medical and therapeutic approach is to treat IgE-mediated diseases. IgE-mediated diseases include atopic diseases (atopic disorders), which are characterized by an inherited propensity to mount an immune response to many common naturally occurring inhaled and ingested antigens, and to produce IgE antibodies continuously. Specific atopic diseases include allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastrointestinal disease. Atopic patients often have multiple allergies, meaning that they have IgE antibodies to, and the symptoms resulting from, many environmental allergens, including pollen, fungi (e.g., molds), animal and insect debris, and certain foods.

However, diseases associated with increased IgE levels are not limited to those with inherited (atopic) etiology. Other diseases associated with increased IgE levels that appear to be IgE-mediated and can be treated with the formulations of the present invention include hypersensitivity reactions (e.g., allergic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis (allergic bronchus aspergillosis), parasitic diseases, interstitial cystitis (intercutial cystitis), hyper-IgE syndrome, ataxia-telangiectasia (ataxia-telangiectasia), Wiskott-Aldrich syndrome, thymic lymphodysplasia (thymic allophonia), IgE myeloma, and graft-versus-host reactions.

Allergic rhinitis, also known as allergic rhinoconjunctivitis or hay fever, is the most common manifestation of atopic reactions to inhaled allergens, the severity and duration of which is often related to the intensity and duration of exposure to the allergen. This is a chronic disease that can first appear at any age, but usually attacks during childhood or adolescence. Typical episodes include profuse watery nasal discharge, paroxysmal sneezing, nasal congestion, and itching of the nose and palate. Mucus drainage behind the nose also leads to pharyngolaryngitis, throat clearing and cough. There may also be symptoms of allergic blepharoconjunctivitis, with intense itching, redness, tearing and photophobia of the conjunctiva and eyelids. Severe episodes are often accompanied by general malaise, weakness, fatigue, and sometimes muscle pain during severe sneezing.

Asthma, also known as reversible obstructive airways disease, is characterized by hyperresponsiveness of the tracheobronchial tree to respiratory stimuli and bronchoconstrictive chemicals, producing episodes of wheezing, dyspnea, chest tightness and coughing that are naturally reversible, or reversible by therapy. This is a chronic disease involving the entire airway, ranging in severity from occasional mild transient reactions to severe, chronic, life threatening bronchial obstruction. Asthma and atopy can coexist, but only about half of asthmatics are also atopy, with a smaller proportion of atopy patients having concurrent asthma. However, atopy and asthma are not completely independent, as asthma occurs more frequently in atopic individuals than in non-atopic individuals, particularly in childhood. Asthma has historically been further broken down into two subgroups, extrinsic asthma and intrinsic asthma.

Extrinsic asthma, also known as allergic, atopic, or immunological asthma, is a description of those patients who typically develop asthma at a young age, usually in the infancy or childhood. Other manifestations of atopic reactions are often co-produced, including eczema or allergic rhinitis. Asthma attacks can occur during the pollen season, in the presence of animals, or when exposed to indoor dust, feather pillows, or other allergens. Skin tests showed positive wheal and erythema response to the causative allergen. Interestingly, total serum IgE concentrations often increase, but sometimes are normal.

Intrinsic asthma, also known as non-allergic or idiopathic asthma (idiopathetic asthma), generally first develops in adulthood, followed by a significant respiratory infection. Symptoms include chronic or recurrent bronchial obstruction not associated with the pollen season or with exposure to other allergens. The test is negative to common atopic allergen skin test, and the serum IgE concentration is normal. Additional symptoms include phlebotomia and eosinophilia. Other protocols that classify asthma into subgroups, like aspirin sensitive, exercise induced, infectious and nevertheless psychological, define external triggers that have a greater impact on some patients than others.

Finally, it is important to note that while some classification approaches have previously only linked allergic asthma to IgE dependence, there is now strong statistically significant data showing a correlation between IgE and asthma (both allergic and non-allergic). Chapter 27, "the Atotic Diseases", A.I.Terr in Medical Immunology, 9th Ed., Simon and Schuster, Stits et al, Ed. (1997). Thus, the term "IgE-mediated disease" for purposes of this application includes allergic and non-allergic asthma.

Signs of an asthma attack include shortness of breath, audible wheezing, and the use of secondary respiratory muscles. There is also generally a rapid pulse and elevated blood pressure, as well as elevated peripheral blood eosinophil levels and increased nasal secretions. Lung function showed a decrease in lung flux and forced expiratory volume at 1 second. Total lung capacity and functional residual capacity are generally normal or slightly increased, but are reduced in extreme bronchospasm.

The pathology of asthma can be distinguished into early and late phase responses. Early stages are characterized by smooth muscle contraction, edema, and hypersecretion, while late reactions are characterized by cellular inflammation. Asthma can be treated by various nonspecific agentsSpecific trigger inducements include infection (e.g., viral respiratory infection), physiological factors (e.g., exercise, hyperventilation, deep breathing, psychological factors), atmospheric factors (e.g., sulfur dioxide, ammonia, cold air, ozone, distilled water vapor), food intake (e.g., propranolol (propranolol), aspirin, non-steroidal anti-inflammatory drugs), experimental inhalants (e.g., hypertonic solutions, citric acid, histamine, methacholine, prostaglandin F_2α) And professional inhalants (e.g., isocyantes). Various other occupational or environmental allergens that cause allergic asthma include animal products, insect dust, marine life, plant products, fruits, seeds, leaves and pollen, organic dyes and inks, microbial agents, enzymes, therapeutic agents, bactericides, inorganic and organic chemicals.

Atopic dermatitis, also known as eczema, neurodermatitis, atopic eczema or Besnier's prurigo, is a common skin disease, characteristic of a patient population with familial and atopic immunological features. The basic characteristic is an inflammatory reaction of the skin with itching, which induces a rash that favors a characteristic symmetrical distribution in certain areas. It is also common for B lymphocytes to overproduce IgE. Although atopic dermatitis is classified as an atopic skin manifestation due to its association with allergic rhinitis and asthma and high IgE levels, the severity of dermatitis is not necessarily associated with experimental exposure of the skin to allergens, and desensitization therapy is not an effective treatment (unlike other allergic diseases). Although high serum IgE levels are a confirmation for diagnosing allergic asthma, normal levels cannot exclude its possibility. Onset of disease can occur at any age, with lesions starting acutely, with erythematous edematous papules or scaly plaques. Itching results in weeping and scabbing, followed by chronic lichenification. At the cellular level, the acute lesions are edematous, with the dermis infiltrated by mononuclear cells, CD4 lymphocytes. Neutrophils, eosinophils, plasma cells and basophils are rare, but degranulated mast cells are present. Chronic lesions are characterized by epidermal hyperplasia, cutaneous hyperkeratosis and parakeratosis, the dermis being infiltrated by monocytes, langerhans cells and mast cells. There may also be fibrosis in the focal region, including the perineurium, which involves the small nerves.

Allergic gastrointestinal disease, also known as eosinophilic gastroenteropathy (eosinophilic gastroenteropathy), is a rare manifestation of atopy, in which multiple IgE food sensitivity is associated with local gastrointestinal mucosal reactions. Is rare in adults, but is more common, but transient, and is present in infants. This occurs when ingested food allergens react with local IgE antibodies on the jejunal mucosa to release mast cell regulators, resulting in digestive system symptoms shortly after a meal. Chronic inflammation resulting from sustained exposure leads to gastrointestinal protein loss and hypoproteinic edema. There may be significant enough blood loss through the inflamed intestinal mucosa to cause iron deficiency anemia. Allergic reactions occur locally in the upper gastrointestinal mucosa after exposure to allergens, but can be relieved by avoiding allergens.

Allergic reactions and urticaria are apparently IgE-mediated, but are not genetically determined and have no preference for atopic individuals. Allergic reactions are systemic allergic reactions that are acute and involve several organ systems simultaneously, usually the cardiovascular, respiratory, skin and gastrointestinal systems. The reaction is immune-mediated, and occurs when exposed to an allergen to which the patient has previously been sensitized. Urticaria and angioedema refer to physical swelling, erythema and itching, caused by histamine-stimulated receptors on the epidermal vessels, which are the hallmark skin features of systemic hypersensitivity. Systemic allergies are IgE-mediated reactions that occur in multiple organs simultaneously, caused by drugs, insect venom or food. It is suddenly caused by allergen-induced IgE loaded on mast cells, resulting in profound and life-threatening changes in the function of various vital organs. Vascular collapse (collapse), acute airway obstruction, cutaneous vasodilation and edema, and gastrointestinal and genitourinary muscle spasms occur almost simultaneously, although not necessarily to the same extent.

The symptoms of anaphylaxis include angioedema and lung hyper-distension, mucus plugs with airways and limited lung atelectasis. At the cellular level, the lung behaves similarly to that during an acute asthma attack, with hypersecretion of the submucosal glands of the bronchi, edema of the mucous membranes and submucosa, congestion of the blood vessels around the bronchi and eosinophilia in the bronchial wall. Pulmonary edema and hemorrhage may be present. Bronchomuscular spasms, hyper-distension, and even alveolar rupture may also be present. Important features of allergic reactions in humans include edema, vascular congestion, and eosinophilia in the lamina propria of the larynx, trachea, epiglottis and tongue.

Exposure to the allergen may be via ingestion, injection, inhalation, or contact with the skin or mucosa. The reaction starts within seconds or minutes after exposure to the allergen. There may be an initial fear or sensation of dying followed by a rapid appearance of symptoms in one or more target organ systems: cardiovascular, respiratory, dermal and gastrointestinal systems.

The allergens responsible for the allergic reaction are different from those commonly associated with atopy. Food, drugs, insect venom or latex (latex) are common sources. Food allergens include those in crustaceans, mollusks (e.g., lobster, shrimp, crab), fish, legumes (legume) (e.g., peanut, pea, broad bean, licorice), seeds (e.g., sesame, cottonseed, caraway, meson, linseed, sunflower), nuts, berries, proteins, buckwheat, and milk. Drug allergens include those allergens found in heterologous proteins and polypeptides, polysaccharides, and hapten drugs. Insect allergens include hymenopteran insects including bees, wasps, hornets, wasps and fire ants.

Epinephrine is the typical medical and metallurgical means for allergic reactions, and antihistamines or other histamine blockers are generally prescribed for less severe urticaria or angioedema reactions.

F. Combination therapy

The methods of the invention may be combined with known methods of treating IgE-mediated diseases, either as a combined or additional therapeutic step, or as an additional component of a therapeutic formulation.

For example, antihistamines, particularly non-sedating antihistamines, can be administered prior to, or in proportion to, the anti-IgE antibodies of the invention. Suitable antihistamines include those belonging to the class of alkylamines (e.g., clofenamide), ethanolamines (e.g., diphenhydramine), and phenothiazines (e.g., pullulan). Many antihistamines antagonize the pharmacological effect of histamine by blocking its receptor site on effector cells, and other common antihistamines act by blocking histamine release from mast cells that have been sensitized or armed with allergen-specific IgE (e.g., cromolyn sodium). Examples of antihistamines include astemizole, pipheptidine maleate, bronhenimine maleate, chlorpyrilamine maleate, cetirizine hydrochloride, clemastine fumarate, cyproheptadine hydrochloride, dexbrompheniramine maleate, dextrochlorpheniramine maleate, chlorophylline diphenhydramine hydrochloride, ducrehydramine succinate, fexofentadine hydrochloride, terphenamine hydrochloride, hydroxyzine hydrochloride, loratidine hydrochloride, tripelennamine citrate, tripelennamine hydrochloride, and propiverine hydrochloride.

Specific symptoms of IgE-mediated diseases (e.g., early stage response) can be ameliorated with sympathomimetic agents or drugs having broncho-diastolic effects. Epinephrine is a widely used alpha and beta adrenergic drug, often administered subcutaneously as 0.2-0.5mL of a 1: 100 aqueous solution. Where a longer duration of effect is desired, a long acting form of epinephrine (e.g., metaproterenol) may also be used in a 1: 200 suspension. Other suitable betaadrenergic agents include salbutamol, pirbuterol, metaproterenol, salmeterol, neoisoprenaline, and formoterol, administered nasally (e.g., a hand-held nebulizer, an intermittent positive pressure breathing device, or a metered dose pressurized inhaler) or orally.

Bronchodilation can also be achieved by administration of xanthine, in particular by co-administration with sympathomimetic drugs as described above. Examples of xanthines include aminophylline (intravenous, 250-500mg) and theophylline (oral, 10-20 μ g/ml serum concentration).

Other symptoms from various IgE-mediated diseases (e.g., late stage reactions) can be attenuated by treatment with glucocorticoids or other drugs with anti-inflammatory effects. Dehydrocortisone (30-60mg per day) was administered systemically for severe episodes, while for long-term maintenance therapy, chlorodimyride dipropionate, fluroxyphyllocortisone acetonide and flunisolide were administered in aerosol form. Other steroids with anti-inflammatory effects include: betamethasone, budesonide, dexamethasone, fludrocortisone acetate, flunisolide, fluticasone propionate, hydrocortisone, methylprednisolone, prednisolone, hydrocortisone, and fluroxypendrol.

Non-steroidal anti-inflammatory drugs that may also be used in combination with the treatment methods of the present invention include acetaminophen, acetylsalicylic acid, bromfenac sodium, diclofenac, diflunisal, etodolac, phenoxyhydridocusate calcium, flurbiprofen, ibuprofen, indochloroformyl, ketoprofen, meclofenamate sodium, mefenamic acid, nabumetone, naproxen sodium, oxyphenbutazone, phenylbutzone, piroxicam, sulindac, tolmetin sodium.

In addition, decongestants (e.g., phenylephrine, phenylpropanolamine, pseudoephahandrin), cough suppressants (e.g., methaphen, codeine, or hydrocodone), or analgesics (e.g., acetaminophen, aspirin) may also be administered to maximize therapeutic benefit.

Allergen desensitization is a form of treatment in which an allergen is injected into a patient to reduce or eliminate the allergic reaction. This is also referred to as allergen immunotherapy, desensitization or allergy injection treatment. It is often used in combination with other allergy treatments, but is often not the primary treatment. It has been successfully applied in situations where allergen avoidance is not possible. Typical allergen desensitization treatments include subcutaneous injections of sterile allergen at increasing doses once or twice a week until a dose is able to produce transient minimal local inflammation at the injection site. This dose is then administered every 2-4 weeks according to a maintenance schedule. Allergic desensitization is most commonly used in the treatment of allergic asthma and allergic rhinitis, although it has also been successful in treating allergic reactions. Desensitization is also effectively achieved by the use of an adjuvant, such as Freund's incomplete adjuvant, which is an emulsion of aqueous antigen in mineral oil. The physiological effect of this creates a depot of undissolved liquid from which droplets of allergen can be gradually released. Another form of allergen desensitization is the polymerization of monomeric allergens with glutaraldehyde to produce molecules of relatively low allergenicity (e.g., leading to allergic reactions) while maintaining effective levels of immunogenicity.

G. The dosage of the medicine is as follows:

the dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the particular use envisioned. Determining the appropriate dosage or route of administration is well within the capability of the ordinarily skilled artisan. Animal testing provides reliable guidance for determining effective dosages for human therapy. Interspecies conversions of effective doses can be performed according to The principles set forth In Mordenti, J. and Chappell, W. "The Use of intersections Scaling In Autocokineticics," In Autocokineticics and New Drug Development, Yacobi et al, Eds, Pergamon Press, New York 1989, pp.42-46.

When performing in vivo administration of the polypeptides or antibodies described herein, the number of normal doses may range from about 10ng/kg up to about 100mg/kg of mammalian body weight or more per day, preferably from about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance on specific dosages and methods of administration has been provided in the literature, see, e.g., U.S. patent nos. 4,657,760; 5,206,344 or 5,225,212. It is within the scope of the present invention that different formulations are effective for different medical and different diseases, and that administration intended to treat a particular organ or tissue may necessarily be administered in a different manner than to treat another organ or tissue. In addition, dosing may be by one or more separate administrations, or by continuous infusion. For repeated administration lasting several days or longer, depending on the conditions thereof, the medical treatment is continued until the desired suppression of the disease symptoms occurs. However, other dosing regimens are also useful. The progress of the treatment can be readily monitored by conventional techniques and analysis.

H. Administration of the formulations

The formulations of the present invention, including but not limited to reconstituted formulations, are administered to a mammal, preferably a human, in need of protein treatment according to known methods, for example intravenously in bolus form (bolus) or by continuous infusion over a period of time by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-synovial, intrathecal, oral, topical or inhalation routes.

In a preferred embodiment, the formulation is administered to the mammal by subcutaneous administration (e.g., under the skin). For this purpose, the formulation can be injected using a syringe. However, other devices for administering the formulation are available, such as injection devices (e.g., injection-ease)^TMAnd Genject^TMA device); injection pen (e.g. GenPen)^TM) (ii) a Automatic injection device, needleless device (e.g. mediJector)^TMAnd BioJector^TM) And subcutaneous patch delivery systems (subcutaneous patch delivery systems).

In a particular embodiment, the invention relates to a kit for single dose administration of the units. Such kits include containers of aqueous formulations of therapeutic proteins or antibodies, including single or multi-chambered pre-filled syringes. An exemplary pre-filled syringe is available from Vetter GmbH, Ravensburg, Germany.

The appropriate dosage of the protein ("therapeutically effective amount") will depend, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous therapy, the patient's history and response to the protein, the type of protein used, and the judgment of the attending physician. The protein may be administered to the patient at once or in a series of medical and metallurgical procedures, or may be administered to the patient at any time after the diagnosis. The protein may be administered as the only medical or metallurgical method, or in combination with other drugs or treatments useful in treating the current condition.

The candidate protein is an antibody, and about 0.1-20mg/kg is the initial candidate dose to be administered to a patient, e.g., whether administered in one or more separate administrations. However, other dosing regimens are also useful. The progress of the treatment can be easily monitored by conventional techniques.

Uses of anti-IgE formulations (e.g., rhuMAbE-25, rhMAbE-26, Hu-901) include the treatment or prevention of, for example, IgE-mediated allergic diseases, parasitic infections, interstitial cystitis (Intersational cystitis), and asthma. Depending on the disease or condition to be treated, a therapeutically effective amount (e.g., about 1-15mg/kg) of the anti-IgE antibody is administered to the patient.

I. Article of manufacture

In another embodiment of the invention, an article of manufacture comprising the formulation is provided, preferably with instructions for its use. The article comprises a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (e.g., single or dual chamber syringes), and test tubes. The container may be made of various materials such as glass or plastic. The container containing the formulation and the label on or associated with the container may indicate instructions for reconstitution and/or use. The label may further indicate that the formulation is useful for or intended for subcutaneous administration. The container with the formulation may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (e.g., BWFI). The final protein concentration in the reconstituted formulation is typically at least 50mg/ml when the diluent and lyophilized formulation are mixed. The article of manufacture may further comprise other desirable materials from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention. All of the contents of this citation are herein incorporated by reference in their entirety.

In another embodiment, the present invention provides an article of manufacture comprising a formulation as described herein for administration in an autoinjector device. An auto-injector may be described as an injection device that delivers its contents upon activation without the additional necessary action of the patient or administrator. They are particularly suitable for self-medication of therapeutic formulations when the administration rate must be constant and the administration time is relatively long.

Example 1

Preparation of an anti-IgE rhuMAbE25 ("E25") formulation

Preparations of monoclonal anti IgE antibody rhuMAbE25 were prepared from E25 bulk (bulk) Lot K9094A (40mg/ml rhuMAb E25, 85mM trehalose, 5mM histidine, pH6, 0.01% Tween 20) or rhuMAbE25Q-Pool (5mg/ml rhuMAb E25, 25mM Tris, 200mM NaCl).

Aqueous solutions of rhuMAbE25 were prepared by dialysis at 2-8 ℃ into different buffers (20mM His-HCl and 200mM Arg-HCl, pH 6.0) using Slide-A-Lyzer dialysis cartridges (Pierce). The sample was then transferred to the sample reservoir of a Centricon-30 centrifugal microconcentrator (Amicon). The protein was concentrated by rotating the Centricon-3 concentrator at 4000-.

The samples were then concentrated to-150 mg/ml rhuMAb E25 using ultrafiltration. Tween 20 was added to each preparation to a final concentration of 0.02%. All formulations were filtered, aseptically filled into 3cc FormaVitrem vials in a Class 100 room, stoppered with 13-mM Diakyo stoppers.

Example 2

The method and the material are as follows:

stability study: all formulations were filled in 1ml volumes into 3 ccFormaVirum glass vials in a Class 100 sterile filling room and stoppered with 13-mm Diakyo stoppers. The vials were placed in light tight containers at-70 deg.C, 2-8 deg.C, 15 deg.C and 30 deg.C.

Agitation study: aliquots of each formulation were placed in glass vials. The vials were agitated horizontally at room temperature on a Glas-Col Bench Top shaker. The shaker was set to 70, with an arm length of 30cm (maximum). After agitation, the samples were examined and analyzed according to the following protocol.

Freeze-thaw study: samples of E25 were subjected to three freeze-thaw cycles. Each cycle included freezing overnight at-70 ℃, and subsequent thawing at room temperature for about one hour. After each cycle, the samples were visually inspected using a light box (light box) to assess the color and transparency of the liquid. Turbidity and soluble aggregates were measured following the protocol described below.

The analysis method comprises the following steps: analysis of sample stability by the methods listed in Table 1

Table 1: analytical method

^a Inspection of color, appearance and clarity：

The color, appearance and clarity of the samples were assessed visually against white and black backgrounds for examination, as compared to an equal volume of negative control. The sample should be carefully swirled to ensure homogenous mixing, but not so much force that bubbles are generated.

^b Size exclusion chromatography：

A TSK SUPER SW3000 (4.6X 300mm) column was used in the HP 1100 chromatography system. The column was loaded with 20. mu.g of protein and eluted in 0.1M potassium phosphate, pH 6.8. The sample was detected with a UV detector at 280 nm.

^c Hydrophobic Interaction Chromatography (HIC)：

HIC experiments were performed on an HP 1100 liquid chromatography system using a TSK Phenyl-5PW (7.5X 75mm) column (TosoHaas). The column was loaded with 28. mu.g papain digested Fab fragments and eluted from 2M to 0M with a concentration gradient of ammonium sulfate in 20mM Tris buffer. The peak was monitored by a UV detector at 210 nm.

^d Turbidity of the mixture：

The turbidity of the samples was determined in a cuvette of 1cm clear length using an HP spectrophotometer. The haze was calculated as the average absorbance at 340-.

^eThe activity of anti-IgE monoclonal antibodies was determined by a receptor binding inhibition assay. Samples were diluted to within a standard curve of 100 and 1.56 μ g/ml in assay diluent containing phosphate buffer, 0.5% BSA, 0.05% polysorbate 20, 0.01% Thimerosol. The microtiter plates were coated with the IgE receptor and then incubated with IgE-biotin and diluted anti-IgE samples. The amount of receptor-bound IgE-biotin associated with the activity of the anti-IgE monoclonal antibody was determined using streptavidin-HRP. The data were analyzed using a 4-parameter logistic curve fitting program.

^fThe concentration of the antibody was obtained on a Hewlett Packard 8453 double-row spectrophotometer using a 1cm quartz cuvette. Using 1.5cm^-1(mg/ml)^-1The concentration was calculated from the absorbance of (b).

Outline of liquid preparation

Stability data of 150mg/ml E25 in histidine and ArgHCl formulations

Stability data for 80mg/ml E25 in histidine and trehalose formulations

a. Size exclusion chromatography to measure soluble aggregates and fragments

b. Hydrophobic interaction chromatography of papain-digested E25

IgE receptor binding inhibition assay

d. Average OD value (340-

Agitation study:

preparation 1: 156mg/ml E25, 200 mMAGRGCl, 23mM His, 0.02% T20

Preparation 2: 150mg/ml E25, 182 mMAGRGCl, 20mM His, 0.02% T20

Freeze-thaw study:

preparation 1: 156mg/ml E25, 200 mMAGRGCl, 23mM His, 0.02% T20

Preparation 2: 150mg/ml E25, 182 mMAGRGCl, 20mM His, 0.02% T20

Example 3

Samples of liquid formulations of anti-IgE monoclonal antibody (E26) were prepared in 20mM buffer and then stored at 30 ℃ and 40 ℃. The stability of E26 was determined by chromatography and activity measurements. Soluble aggregates were determined by size exclusion chromatography and isomerization was measured by hydrophobic interaction chromatography on pepsin digested samples. The activity of the samples was monitored using an IgE receptor binding inhibition assay. As shown in fig. 1, 2 and 3, the degradation of E26 is highly dependent on the pH of the buffer. E26 appeared most stable around pH 6.0.

Example 4

Microparticle formulation is a major challenge in making high concentration liquid formulations because it generally increases with increasing protein concentration under stress conditions. Figure 4 shows the results of an agitation study on a concentrated E26 liquid formulation. Formulations were prepared in 20mM succinate, 192mM trehalose, pH6.0, and varying concentrations of polysorbate 20. The microparticle formulation was monitored by turbidity measurements. The results show that the turbidity of the E26 solution increases with agitation time. Addition of at least 0.01% polysorbate under stress is essential to reduce microparticle formation. Similar results were observed for the concentrated E25 liquid formulation.

Example 5

Figure 5 shows a liquid formulation of 150mg/ml E25 prepared by reconstitution of lyophilized E25. An increase in salt concentration inhibits reversible microparticle formation and results in a decrease in turbidity readings. Of all the salts tested, the formulation containing Arg-HCl appeared to have the least turbidity. The effect of salt concentration on reducing turbidity readings was also observed for E25 prepared by the TFF method.

Example 6

The E25 liquid formulation in the presence of ArgHCl also appears to have better stability than other liquid formulations. FIGS. 6 and 7 show the reaction between ArgHCl and CaCl₂And MgCl₂The liquid formulation of (2) has the result of stability study of E25 at 150 mg/ml. For liquid formulations comprising ArgHCl, with or without sucrose, there was a slight difference in their stability with respect to turbidity, isomerization and fragmentation. Liquid formulation comprising ArgHCl to MgCl₂And CaCl₂The liquid preparation of (3) is more stable.

Example 7

Figure 8 shows the results of stability studies for acetate containing E25 liquid formulations and histidine formulations. Histidine containing formulations have a higher pH than acetate formulations. The results clearly show that E25 is more stable in histidine, ArgHCl liquid formulations than under other conditions.

Example 8

High concentrations of E25 can form solid gels in the presence of certain ions, such as citrate, succinate and sulfate (table 1), especially at storage temperatures of 2-8 ℃. The use of arginine-HCl as an excipient allowed us to formulate E25 to exceed 200mg/ml without gel or precipitate formation.

Table 1: effect of various excipients on gelation of E25 at 125mg/ml, pH6.0

Example 9

Expression of proteins or antibodies in Escherichia coli

This example illustrates the preparation of a non-glycosylated form of a desired protein or antibody by recombinant expression in Escherichia coli.

Initially, DNA sequences encoding the desired protein or antibody are amplified using selected PCR primers. The primer should contain a restriction enzyme site that corresponds to the restriction enzyme site on the selected expression vector. Various expression vectors can be used. An example of a suitable vector is pBR322 containing ampicillin and tetracycline resistance genes (from Escherichia coli; see Bolivar et al, Gene,2: 95(1977)). The vector was digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into a vector. The vector preferably comprises a sequence encoding an antibiotic resistance gene, a trp promoter, a polyhis leader (comprising the first six STII codons, a polyhis sequence and an enterokinase cleavage site), a coding region for the desired protein or antibody, a lambda transcription terminator and the argU gene. In addition, the vector may include at least non-translated 5 'and 3' portions of the native nucleic acid sequence encoding the desired protein or antibody.

The selected Escherichia coli strain is then transformed with the ligation mixture using the methods described in Sambrook et al, supra. Transformants were identified by their ability to grow on LB plates and antibiotic resistant colonies were selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

The selected clones may be grown overnight in liquid medium, e.g. in LB broth supplemented with antibiotics. The overnight culture can then be used to inoculate larger scale cultures. The cells are then grown to the desired optical density, at which time the promoter is expressed openly.

After several more hours of culturing the cells, the cells can be harvested by centrifugation. The cell pellet obtained by centrifugation is lysed with various reagents known in the art, and the lysed desired protein or antibody can then be purified using a metal chelating column under conditions that allow for tight binding of the protein or antibody.

The desired protein or antibody can be expressed in poly-His tagged form in Escherichia coli using the following procedure. Initially, DNA sequences encoding the desired protein or antibody are amplified using selected PCR primers. The primers contain restriction enzyme sites corresponding to those on the selected expression vector, as well as other useful sequences that provide for efficient and reliable translation initiation, rapid purification on metal chelating columns, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequence was then ligated into an expression vector for transformation of the strain 52-based Escherichia coli host (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq.) the transformants were first cultured in LB containing 50mg/mL carbenicillin with shaking at 30 ℃ until 3-5 O.D.600 was reached and then the culture was diluted 50-100 fold to CRAP medium (by mixing 3.57g (NH) in 500mL water₄)₂SO₄0.71g sodium citrate $2H2O, 1.07g KCl, 5.36g Difco yeast extract, 5.36g Sheffield ghost SF, and 110mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7mM MgSO₄Prepared) and grown at 30c for about 20-30 hours with shaking. Samples were taken and analyzed by SDS-PAGE to confirm expression, and bulk cultures were centrifuged to pellet the cells. The cell pellet was frozen until initial purification and refolding were performed.

Escherichia coli (6-10g cell pellet) obtained from 0.5 to 1L of the fermentation broth was resuspended in 10 times (w/v) of 7M guanidine, 20mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate were added to give final concentrations of 0.1M and 0.02M, respectively, and the solution was stirred at 4 ℃ overnight. This procedure results in a denatured protein with all cysteine residues blocked by sulfitation. The solution was centrifuged in a Beckmann Ultracentifige for 30 minutes at 40,000 rpm. The supernatant was diluted with 3-5 volumes of metal chelating column buffer (6M guanidine, 20mM Tris, pH 7.4) and clarified by filtration through a 0.22 micron filter. The clarified extract was loaded onto a 5ml Qiagen Ni-NTA metal chelating column equilibrated in metal chelating column buffer. The column was washed with additional buffer containing 50mM imidazole (Calbiochem, Utrol grade) pH 7.4. The protein was eluted with a buffer containing 250mM imidazole. Fractions containing the desired protein were collected and stored at 4 ℃. The protein concentration was estimated by absorbance at 280nm using an extinction coefficient calculated from its amino acid sequence.

Proteins were refolded by slowly diluting the samples into freshly prepared refolding buffer consisting of: 20mM Tris, pH 8.6, 0.3M NaCl, 2.5M urea, 5mM cysteine, 20mM glycine and 1mM EDTA. The refold volume was chosen so that the final protein concentration was between 50-100 micrograms/ml. The refolding solution was stirred gently at 4 ℃ for 12-36 hours. The refolding reaction was terminated by adding TFA to a final concentration of 0.4% (pH about 3). Prior to further protein purification, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1/H reverse phase column using a mobile buffer of 0.1% TFA, eluting with a gradient from 10% to 80% acetonitrile. Aliquots of the fractions with a280 absorbance were analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein were collected. Generally, most properly refolded species of proteins elute at the lowest acetonitrile concentration, as these folded protein species are the most compact, with their hydrophobic interior masked from interaction with the reverse phase resin. Aggregated species typically elute at higher acetonitrile concentrations. In addition to removing the misfolded form of the protein from the desired form of the protein, the reverse phase step also removes endotoxin from the sample.

The fractions containing the folded desired protein or antibody were collected and acetonitrile was removed using a gentle stream of nitrogen in solution. The protein was formulated as 20mM Hepes, pH 6.8, in a solution containing 0.14M sodium chloride and 4% mannitol, and sterile filtered, either by dialysis or by gel filtration using G25Superfine (Pharmacia) resin equilibrated in formulation buffer.

Example 10

Expression of proteins or antibodies in mammalian cells

This example illustrates the preparation of a potentially glycosylated form of a desired protein or antibody by recombinant expression in mammalian cells.

Vector pRK5 (see EP 307,247 published on 3/15/1989) was used as an expression vector. Optionally, DNA encoding the desired protein or antibody is ligated into pRK5 using selected restriction enzymes to allow insertion of such DNA using ligation methods such as those described in Sambrook et al, supra.

In one embodiment, the host cell of choice may be a 293 cell. Human 293 cells (ATCCCCL 1573) are grown to confluence on tissue culture plates in medium, e.g. DMEM supplemented with fetal bovine serum and optionally nutrients and/or antibiotics. About 10. mu.g of DNA encoding the desired protein or antibody linked to pRK5 was combined with about 1. mu.g of DNA encoding the VA RNA gene [ Thimmappaya et al, Cell,31：543(1982)]the DNA of (4) was mixed and dissolved in 500. mu.l of 1mM Tris-HCl, 0.1mM EDTA, 0.227M CaCl₂In (1). To this mixture was added dropwise 500. mu.l of 50mM HEPES (pH 7.35), 280mM NaCl, 1.5mM NaPO₄The precipitate was allowed to form for 10 minutes at 25 ℃. The pellet was suspended, added to 293 cells and allowed to settle for about four hours at 37 ℃. The medium was aspirated off and 2ml of PBS containing 20% glycerol was added for 30 seconds. The 293 cells were then washed with serum-free medium, fresh medium was added, and the cells were incubated for about 5 days.

At about 24 hours post-transfection, the medium was removed and replaced with medium (separately)) Or comprises 200. mu. Ci/ml³⁵S-cysteine and 200. mu. Ci/ml³⁵A culture medium of S-methionine. After 12 hours of incubation, conditioned media were collected, concentrated on rotary filters, and loaded onto 15% SDS gels. The processed gel may be dried and exposed to the film for a selected period of time to reveal the presence of the desired protein or antibody. The culture containing the transfected cells may be subjected to further incubation (in serum-free medium) and the medium tested in the selected biological detection method.

In an alternative technique, Somparyrac et al, Proc. Natl. Acad. Sci, is used.12: 7575(1981), the desired protein or antibody can be transiently introduced into 293 cells. 293 cells were grown to maximum density in rotary flasks and 700. mu.g of DNA encoding the desired protein or antibody ligated into pRK5 was added. Cells were first concentrated by centrifugation from a spinner flask and washed with PBS. The DNA-dextran pellet was incubated on the cell pellet for four hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-added to spinner flasks containing tissue culture medium, 5. mu.g/ml bovine insulin and 0.1. mu.g/ml bovine transferrin. After about four days, the conditioned medium was centrifuged and filtered to remove cells and debris. The sample containing the expressed desired protein or antibody may then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, the desired protein or antibody may be expressed in CHO cells. Known reagents may be used, for example CaPO₄Or DEAE-dextran, DNA encoding the desired protein or antibody linked to pRK5 was transfected into CHO cells. As described above, the cell culture may be incubated, the medium replaced with medium (alone) or contain a radioactive label, e.g.³⁵A culture medium of S-methionine. After the presence of the desired protein or antibody is determined, the culture medium can be replaced with serum-free medium. Preferably, the culture is incubated for about 6 days and then the conditioned medium is harvested. The medium containing the expressed desired protein or antibody may then be concentrated and purified by any selected method。

Epitope-tagged variants of the desired protein or antibody may also be expressed in host CHO cells. DNA encoding the desired protein or antibody linked to pRK5 can be subcloned from pRK5 vector. The subcloned inserts may be PCR fused in-frame to a selected epitope tag, such as a poly-his tag, into a baculovirus expression vector. Poly-his labeled DNA encoding the desired protein or antibody insert may then be subcloned into an SV40 driven vector containing a selection marker for selecting stable clones, such as DHFR. Finally, CHO cells (as described above) can be transfected with the SV 40-driven vector. The expression can be confirmed by labeling as described above. This can then be done by any chosen method, e.g. Ni²⁺Chelate affinity chromatography to concentrate and purify the medium containing the expressed poly-His-tagged desired protein or antibody.

The desired protein or antibody may also be expressed in CHO and/or COS cells by a transient expression step, or in CHO cells by another stable expression step.

Stable expression in CHO cells was performed using the following procedure. The proteins are expressed as IgG structures (immunoadhesins) in which the coding sequence for the soluble form (e.g., the extracellular region) of the respective protein is fused to an IgG1 constant region sequence comprising a hinge region, CH2 and CH2 regions, and/or is in the form of a poly-His tag.

After PCR amplification, each DNA was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al, Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed with restriction sites 5 ═ and 3 ═ compatible with the DNA of interest to allow convenient movement of the cDNA. Vectors used in CHO cell expression such as Lucas et al, Nucl.24: 9(1774-1779(1996), the SV40 early promoter/enhancer was used to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression allowed selection of cells stably maintained the plasmid after transfection.

Using the commercial transfection reagent Superfect^TM(Quiagen)、Dosper^TMOr Fugene^TM(Boehringer Mannheim) twelve micrograms of the desired plasmid DNA were introduced into approximately 1000 ten thousand CHO cells. Cells were grown as described in Lucas et al, supra. Will be about 3X 10^-7Cells were frozen in ampoules for further growth and production as described below.

The ampoule containing the plasmid DNA was thawed by placing in a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10mL of media and centrifuged at 1000rpm for 5 minutes. The supernatant was aspirated and the cells resuspended in 10mL selection medium (containing 5% 0.2)0.2 of filtered fetal bovine serumFiltered PS 20). The cells were then aliquoted into a 100mL spinner containing 90mL of selection medium. After 1-2 days, cells were transferred to a 250mL spinner filled with 150mL of selective growth medium and incubated at 37 ℃. After 2-3 days, the ratio is 3X 10⁵cells/mL seeded 250mL, 500mL, and 2000mL spinners. The cell culture medium was replaced with fresh medium by centrifugation and resuspension in production medium. Although any suitable CHO medium can be used, in practice the production medium described in U.S. Pat. No.5,122,469, published 16.6.1992, can be used. At 1.2X 10⁶Individual cells/mL were seeded with a 3L production spinner. At day 0, cell number and pH were determined. On day 1, the spinner was sampled and aeration with filtered air was initiated. On day 2, the spinner was sampled, the temperature was changed to 33 ℃ and 30mL of 500g/L glucose and 0.6mL of 10% antifoam (e.g., 35% dimethicone emulsion, Dow Coming 365 medical grade emulsion) were added. During the whole production process, the pH was adjusted as necessary to maintain around 7.2. After 10 days, or until the survival rate drops below 70%, by centrifugation and passage through 0.22The cell culture was harvested by filtration through a filter. The filtrate can be stored at 4 ℃ or immediately loaded onto a column for purification.

For the poly-His tagged structure, the protein was purified using a Ni-NTA column (Qiagen). Imidazole was added to the conditioned medium to a concentration of 5mM prior to purification. Conditioned medium was pumped at a flow rate of 4-5ml/min onto a 6ml Ni-NTA column equilibrated in 20mM Hepes, pH 7.4 buffer containing 0.3M NaCl and 5mM imidazole. After loading, the column was washed with additional equilibration buffer, and the protein was eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was then desalted using a 25ml G25Superfine (Pharmacia) column, placed in storage buffer containing 10Hepes, 0.14MNaCl and 4% mannitol, pH 6.8, and stored at-80 ℃.

Immunoadhesin (Fc containing) structures were purified from conditioned media as follows. Conditioned medium was pumped onto a 5ml protein A column (Pharmacia) equilibrated with 20mM Na phosphate buffer, pH 6.8. After loading, the column was carefully washed with equilibration buffer and then eluted with 100mM citric acid, pH 3.5. By collecting 1ml fractions to a fraction containing 275Eluted proteins were immediately neutralized in 1M Tris buffer, pH 9 tubes. The highly purified protein was then desalted and placed in storage buffer for poly-His protein as described above. Homogeneity (homogeneity) was assessed by SDS polyacrylamide gel and N-terminal amino acid sequencing by Edman degradation.

Example 11

Expression of antibodies or proteins in yeast

The following methods describe recombinant expression of the desired protein or antibody in yeast.

First, a yeast expression vector is constructed to produce or secrete a desired protein or antibody intracellularly from the ADH2/GAPDH promoter. The DNA encoding the desired protein or antibody and promoter are inserted into appropriate restriction enzyme sites in the selected plasmid for direct intracellular expression. For secretion, DNA encoding the desired protein or antibody is cloned into a selected plasmid, along with DNA encoding the ADH2/GAPDH promoter, a native signal peptide or other mammalian signal peptide, or, for example, yeast alpha factor or invertase secretion signal/leader sequence, and a linker sequence (if necessary), for expression of the desired protein or antibody.

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in the selected fermentation medium. Analysis can be performed by precipitation of the transformed yeast supernatant with 10% trichloroacetic acid, separation by SDS-PAGE, staining of the gel with Coomassie blue.

The yeast cells are subsequently removed from the fermentation medium by centrifugation, and the medium is then concentrated using selected cartridge filters to isolate and purify the recombinant protein or antibody. The concentrate containing the recombinant protein or antibody may be further purified using a selected column chromatography resin.

Example 12

Expression of proteins or antibodies in baculovirus infected insect cells

The following methods describe recombinant expression of the desired protein or antibody in baculovirus-infected insect cells.

A sequence encoding the desired protein or antibody is fused upstream of the epitope tag contained in the baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like the Fc region of IgG). Various plasmids can be used, including those derived from commercial sources, such as pVL1393 (Novagen). Briefly, the sequences encoding the desired portion of the protein or antibody, e.g., the extracellular region of a transmembrane protein, or the mature protein if the protein is an extracellular protein, are amplified by PCR using primers complementary to the 5 'and 3' regions. The 5' primer may include flanking (selected) restriction endonuclease sites. The product is then digested with those selected restriction enzymes and subcloned into an expression vector.

The above plasmid and Baculogold were prepared by using lipofectin (commercially available from GIBCO-BRL)^TMViral dna (pharmingen) was co-transfected into Spodoptera frugiperda ("Sf 9") cells (ATCCCRL 1711) to produce recombinant baculovirus. After incubation at 28 ℃ for 4-5 days, the released virus was harvested for further amplification. Such as O' Reilley et al, Bacillus expression vectors: a laboratory manual, Oxford: oxford University Press (1994) describes viral infection and protein expression.

Then may be passed through Ni, for example²⁺The expressed poly-his-tagged protein or antibody is purified by chelate affinity chromatography. Such as the Rupert et al, Nature,362: 175-179(1993) describe the preparation of extracts from recombinant virus-infected Sf9 cells. Briefly, Sf9 cells were washed and resuspended in sonication buffer (25mL Hepes, pH 7.9; 12.5mM MgCl)₂(ii) a 0.1mM EDTA; 10% glycerol; 0.1% NP-40; 0.4M KCl), sonicated twice for 20 seconds on ice. The sonicate was clarified by centrifugation and the supernatant diluted 50-fold in loading buffer (50mM phosphate, 300mM NaCl, 10% glycerol, pH 7.8) over 0.45And (5) filtering by using a filter. Preparation of Ni with a bed volume of 5mL²⁺NTA agarose columns (commercially available from Qiagen), washed with 25mL water, equilibrated with 25mL loading buffer. The filtered cell extract was loaded onto the column at 0.5mL per minute. Wash column to baseline A with Loading buffer₂₈₀At this point fraction collection is started. The column was then washed with a second wash buffer (50mM phosphate; 300mM NaCl, 10% glycerol, pH 6.0) to elute non-specific bindingThe protein of (1). After reaching the a280 baseline again, the column was operated with a gradient of 0 to 500mM imidazole in the second wash buffer. One milliliter fractions were collected and stained with Ni bound to alkaline phosphatase by SDS-PAGE and silver staining or Western blotting²⁺NTA (Qiagen) for analysis. Collecting His containing eluate₁₀The labeled protein or antibody fraction is dialyzed against loading buffer.

Alternatively, purification of IgG-labeled (or Fc-labeled) proteins or antibodies can be performed using known chromatographic techniques, including, for example, protein a or protein G column chromatography.

Example 13

Preparation of antibodies

This example illustrates the preparation of monoclonal antibodies that bind to the protein of interest or the desired antigen.

Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that may be used include purified desired proteins or antibodies of interest, fusion proteins comprising the desired protein or antigen of interest, and cells expressing such recombinant proteins or antigens on the cell surface. The skilled artisan can select an immunogen without undue experimentation.

Mice are immunized with the desired protein or immunogen of interest emulsified in complete Freund's adjuvant by subcutaneous or intraperitoneal injection in amounts of 1-100 micrograms, e.g., Balb/c. Alternatively, the immunogen was emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the hind footpads of the animals. The immunized mice were then boosted 10 to 12 days later with additional (additional) immunogen emulsified in the selected adjuvant. Several weeks thereafter, mice may also be boosted with additional immunization injections. Serum samples are periodically taken from mice by posterior orbital bleeding for detection of antibodies to the desired protein or antigen in an ELISA assay.

After detection of a suitable antibody titer, animals that are "positive" for the antibody can be given a final intravenous injection with the desired protein or antigen of interest. Three to four days later, the mice were sacrificed and splenocytes were harvested. The splenocytes are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line, e.g., p3x63agu.1, available from ATCC, No. crl 1597. The fusion produces hybridoma cells, which can then be placed in 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened for reactivity to the desired protein or antigen of interest using ELISA. It is within the ability of one skilled in the art to determine "positive" hybridoma cells that secrete such monoclonal antibodies.

The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce peritoneal fluid containing such monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Monoclonal antibodies produced in the peritoneal fluid can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of antibodies to protein a or protein G may be used.

Example 14

Purification of desired proteins using specific antibodies

The desired protein may be purified in native or recombinant form by a variety of standard techniques in the art of protein purification. For example, a precursor polypeptide, mature polypeptide, or pre-polypeptide form of a desired protein can be purified by immunoaffinity chromatography using antibodies specific for the desired protein. Generally, immunoaffinity chromatography columns are constructed by covalently attaching antibodies that specifically bind to the desired protein to an activated chromatography resin.

Polyclonal immunoglobulins were prepared from immune sera by precipitation with ammonium sulfate or by purification on immobilized protein a (Pharmacia LKBBiotechnology, Piscataway, n.j.). Similarly, monoclonal antibodies were prepared from mouse peritoneal fluid by ammonium sulfate precipitation or chromatography on immobilized protein a. Covalently attaching partially purified immunoglobulins to chromatography resins, e.g. CnBr-activated Sepharose^TM(Pharmacia LKB Biotechnology). The antibody was coupled to the resin according to the manufacturer's instructions, the resin was blocked, and the derivatized resin was washed.

Such immunoaffinity columns are used to purify the desired protein by preparing fractions from cells expressing the protein in a soluble form. Such preparations are obtained by lysing whole cells or subcellular fractions obtained by differential centrifugation by the addition of detergents or other methods known in the art. Alternatively, a soluble protein comprising a signal sequence can be secreted into the medium in which the cells are grown in an effective amount.

The solubilized preparation comprising the desired protein is passed through an immunoaffinity column, which is washed under conditions that allow preferential absorption of the desired protein (e.g., a high ionic strength buffer in the presence of a detergent). The column is then washed with a buffer at low pH (e.g., about pH 2-3, or a chaotrope at high concentration (e.g., urea or thiocyanate ions) to disrupt binding of the antibody to the protein, and the desired protein is collected.

Claims (10)

1. A stable, low turbidity, liquid formulation comprising (a) a protein or antibody in an amount of 100 to 260mg/ml, (b) arginine-HCl in an amount of 50 to 200mM, (c) histidine in an amount of 10 to 100mM, (d) polysorbate in an amount of 0.01 to 0.1%, wherein the formulation further has a pH of from 5.5 to 7.0, a kinematic viscosity of about 50cs or less, and an osmolality of from 200 to 450 mOsm/kg.

2. A stable, low turbidity liquid formulation comprising (a) an anti-IgE monoclonal antibody in an amount of 100 to 260mg/ml, (b) arginine-HCl in an amount of 50 to 200mM, (c) histidine in an amount of 10 to 100mM, (d) polysorbate in an amount of 0.01 to 0.1%, wherein the formulation further has a pH value of from 5.5 to 7.0, a kinematic viscosity of about 50cs or less, and an osmolality of from 200 to 450 mOsm/kg.

3. A stable, low turbidity liquid formulation comprising (a) an anti-IgE antibody in an amount of about 150mg/ml, (b) arginine-HCl in an amount of 200mM, (c) histidine in an amount of 20mM, (d) polysorbate in an amount of 0.02%, wherein the formulation further has a pH of 6.0.

4. An article of manufacture comprising a container containing the formulation of claim 1.

5. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of claim 3.

6. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof an effective amount of the formulation of claim 3 in combination with an antihistamine.

7. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of claim 3 in combination with a bronchodilator.

8. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of claim 3 in combination with a glucocorticoid.

9. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of claim 3 in combination with the administration of allergen desensitization therapy.

10. A method of treating an IgE-mediated disorder comprising administering to a patient in need thereof a therapeutically effective amount of the formulation of claim 3 in combination with an NSAID.