STABLE FORMULATIONS FOR ANTI-CD19 ANTIBODIES AND ANTIBODY-DRUG CONJUGATES

Abstract

This disclosure provides optimized formulations for CD19 antibodies and antibody-drug conjugates (ADCs)

Claims

1. A stable formulation comprising an anti-CD19 antibody conjugated to monomethylauristatin F (MMAF), a phosphate buffer, a surfactant and a bulking agent, wherein the anti-CD19 antibody comprises a light chain variable region having an amino acid sequence of SEQ ID NO:1 and a heavy chain variable region having an amino acid sequence of SEQ ID NO:2, wherein the phosphate buffer has a pH value between 5.0 and 7.0, wherein the surfactant is polysorbate 80 at a concentration between 0.01% and 0.05% w/v, and wherein the bulking agent is a sugar at a concentration between 10 mg/ml and 75 mg/ml.

2. The stable formulation of claim 1, wherein the phosphate buffer is a sodium phosphate buffer.

3. The stable formulation of claim 1, wherein the phosphate buffer is a potassium phosphate buffer.

4. The stable formulation of claim 1, wherein the phosphate buffer has a pH between about 5.5 and about 6.5.

5. The stable formulation of claim 1, wherein the phosphate buffer has a pH of about 6.0.

6.-11. (canceled)

12. The stable formulation of claim 1, wherein the polysorbate 80 is at a concentration of about 0.02% w/v.

13-14. (canceled)

15. The stable formulation of claim 14, wherein the sugar is selected from the group consisting of sucrose and trehalose.

16. (canceled)

17. The stable formulation of claim 15, wherein the sugar is sucrose.

18. The stable formulation of claim 17, wherein the sucrose concentration is about 60 mg/ml.

19. A stable formulation of claim 1, wherein the phosphate buffer is a potassium phosphate buffer at about pH 6.0.

20. A stable lyophilized anti-CD19 antibody formulation, made by lyophilizing the formulation of claim 19.

21. A vial comprising the stable lyophilized anti-CD19 antibody formulation of claim 20, in an amount for administration to a patient in need of such formulation.

22. A stable liquid anti-CD19 antibody formulation of claim 19.

23. A vial comprising the stable liquid anti-CD19 antibody formulation of claim 22, in an amount for administration to a patient in need of such formulation.

24. The stable formulation of claim 1, wherein the concentration of the anti-CD19 antibody conjugated to monomethylauristatin F (MMAF) is about 15 mg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1A provides an assessment of the stability of the humanized anti-CD19 antibody, hBU12, in various buffers at various pH values.

[0054] FIG. 1B provides an assessment of the stability of the humanized anti-CD19 antibody hBU12 conjugated to MMAF, SGN-CD19A, in various buffers at various pH values.

[0055] FIG. 2 provides an assessment of the effect of pH on the chemical stability of the humanized anti-CD19 antibody hBU12 conjugated to MMAF, SGN-CD19A.

[0056] FIG. 3A demonstrates the effect of freeze/thaw on subvisible particles in hBU12 formulation without polysorbate 80 (0 and 1 freeze/thaw cycle).

[0057] FIG. 3B demonstrates the effect of freeze/thaw on subvisible particles in hBU12 formulation without polysorbate 80 (2 and 3 freeze/thaw cycles).

[0058] FIG. 4 demonstrates the effect of freeze/thaw on subvisible particles in hBU12 formulation with 0.01% polysorbate 80 (0, 1, 2 and 3 freeze/thaw cycles).

[0059] FIG. 5 demonstrates the effect of freeze/thaw on subvisible particles in hBU12 formulation with 0.02% polysorbate 80 (0, 1, 2 and 3 freeze/thaw cycles).

[0060] FIG. 6 demonstrates the effect of freeze/thaw on subvisible particles in hBU12 formulation with 0.03% polysorbate 80 (0, 1, 2 and 3 freeze/thaw cycles).

[0061] FIG. 7 summarizes data on the effect of two sugars, sucrose and trehalose, on the stability of CD19 ADC lyophilized formulations.

[0062] FIG. 8 provides the structure of SGN-CD19A, a CD19 antibody drug conjugate (ADC) comprising the mcMMAF drug linker and the humanized antibody hBU12.

DETAILED DESCRIPTION

[0063] This invention provides stable formulations of anti-CD19 antibodies and of ADC's comprising anti-CD19 antibodies. The formulations include a phosphate buffer, a sugar, and polysorbate-80. The formulations provide stability for the anti-CD19 antibodies and ADC's comprising anti-CD19 antibodies in both liquid and lyophilized embodiments.

[0064] In preferred embodiments, the invention provides a stable formulation of a humanized anti-CD19 antibody, hBU12, as described below. While working with the hBU12 antibody, it was discovered that the hBU12 antibody was unexpectedly unstable and oxidized in the presence of light. An oxidized impurity was detected after incubation of the antibody in the presence of light. Many formulations were tested for their ability to improve the stability of the antibody in the presence of light and to decrease the amount the oxidized impurity. However, the greatest decrease in the oxidized impurity came with use of phosphate buffer in the formulation. The decrease in oxidation associated with light was seen in formulations of unconjugated hBU12 antibody, as well as formulations of the hBU12 antibody conjugated to a cytotoxic agent, for example, mcMMAF.

[0065] Also disclosed are other aspects of the hBU12 formulation that improve the stability of the antibody or antibody drug conjugate. The optimized aspects include, for example, buffer pH, addition of sugars, and addition of surfactants.

Anti-CD19 Antibodies, ADC's, and Formulations

[0066] The pharmaceutical formulations of the present invention comprise a humanized antibody, or an antibody-drug conjugate, that binds specifically to the human CD19 protein. As used herein, the term CD19 means a human CD19 protein or cluster of differentiation 19 protein. The amino acid sequence of human CD19 is known and is disclosed, e.g., at NCBI Reference Sequence: NP_001171569.1. The CD19 protein is a marker for B-cell development and is expressed on B cells at many stages of B cell development.

[0067] In preferred embodiments, the formulations disclosed herein are stable formulations of the SGN-19A therapeutic agent. SGN-CD19A is produced by the conjugation of drug-linker intermediate maleimidocaproyl monomethyl auristatin F (mcMMAF) to the humanized antibody hBU12 (FIG. 8). The points of attachment are cysteines produced by reduction of inter-chain disulfides. SGN-CD19A has an average of four drugs per antibody molecule.

[0068] Methods of making the hBU12 antibody are disclosed, e.g., at U.S. Pat. No. 7,968,687. The amino acid sequence of the light chain variable region of hBU12 is provided herein as SEQ ID NO:1. The amino acid sequence of the heavy chain variable region of hBU12 is provided herein as SEQ ID NO:2. hBU12 is an IgG1 antibody and the variable regions are joined to human heavy and light constant regions. U.S. Pat. No. 7,968,687 also provides methods for the synthesis of mcMMAF and its conjugation to hBU12.

[0069] SGN-CD19A, therefore, is an antibody-drug conjugate (ADC) that delivers mcMMAF to CD19-positive cells. mcMMAF is a tubulin-binding molecule. SGN-CD19A has a proposed multi-step mechanism of action initiated by binding to their target on the cell surface and subsequent internalization. After cell surface binding, internalization, and trafficking of SGN-CD19A through the endocytic pathway, proteolytic degradation of hBU12 in the lysosomes releases the cysteine adduct of the drug linker in the form of cys-mcMMAF, which then becomes available for tubulin binding. See, e.g., Doronina et al., Nat Biotechnol 21:778-84 (2003) and Doronina et al., Bioconjug Chem 17: 114-24 (2006). cys-mcMMAF and mcMMAF are used interchangeably herein. Binding of the released drug to tubulin disrupts the cellular microtubule network, leading to G2/M phase cell cycle arrest and subsequent onset of apoptosis in the targeted cell.

[0070] The antibody component of SGN-CD19A can degrade in the presence of light. The formulations disclosed herein provide improved stability of SGN-CD19A. Moreover, the formulations disclosed herein allow choice of liquid or lyophilized formulations, depending on the needs of the user.

Formulations and Excipients in General

[0071] Excipients are additives that either impart or enhance the stability and delivery of a drug product (e.g., an antibody or ADC). Regardless of the reason for their inclusion, excipients are an integral component of a formulation and therefore need to be safe and well tolerated by patients. For protein drugs, the choice of excipients is particularly important because they can affect both efficacy and immunogenicity of the drug. Hence, protein formulations need to be developed with appropriate selection of excipients that afford suitable stability, safety, and marketability.

[0072] The principal challenge in developing formulations for proteins is stabilizing the product against the stresses of manufacturing, shipping and storage. The role of formulation excipients is to provide stabilization against these stresses. Excipients are also employed to reduce viscosity of high concentration protein formulations in order to enable their delivery and enhance patient convenience. In general, excipients can be classified on the basis of the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to alleviate the effects of a specific stress or to regulate a particular susceptibility of a specific protein. Other excipients have more general effects on the physical and covalent stabilities of proteins. The excipients described herein are organized either by their chemical type or their functional role in formulations. Brief descriptions of the modes of stabilization are provided when discussing each excipient type.

[0073] Given the teachings and guidance provided herein, those skilled in the art will know what amount or range of excipient can be included in any particular formulation to achieve a biopharmaceutical formulation of the invention that promotes retention in stability of the antibody or ADC. For example, the amount and type of a salt to be included in a biopharmaceutical formulation of the invention is selected based on the desired osmolality (i.e., isotonic, hypotonic or hypertonic) of the final solution as well as the amounts and osmolality of other components to be included in the formulation.

[0074] Further, where a particular excipient is reported in molar concentration, those skilled in the art will recognize that the equivalent percent (%) w/v (e.g., (grams of substance in a solution sample/mL of solution)100%) of solution is also contemplated.

[0075] The stability of a pharmacologically active protein formulation is usually observed to be maximal in a narrow pH range. This pH range of optimal stability needs to be identified early during pre-formulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this endeavor. See, e.g., Remmele R. L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999). Once a formulation is finalized, the protein must be manufactured and maintained throughout its shelf-life. Hence, buffering agents are almost always employed to control pH in the formulation. As disclosed above, the hBU12 antibody exhibited improved photostability in the presence of phosphate buffers, as compared to other buffering agents. The optimal pH value for stability was investigated and found to range between pH values of 5.5 and 6.5. In preferred embodiments the pH value is about 6.0. In further embodiments, the pH value is 6.0.

[0076] The phosphate buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level. In one embodiment, the pH buffering concentration is between 0.1 mM and 500 mM (1 M). For example, it is contemplated that the phosphate buffering agent is at least 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM.

[0077] In one aspect of the present pharmaceutical formulations, a stabilizer (or a combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or turbid solution upon reconstitution indicates that the protein has precipitated or at least aggregated. The term stabilizer means an excipient capable of preventing aggregation or physical degradation, including chemical degradation (for example, autolysis, deamidation, oxidation, etc.) in an aqueous state. Stabilizers contemplated include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds, including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid, sodium chloride. See, e.g., Carpenter et al., Develop. Biol. Standard 74:225, (1991). In the present formulations, the stabilizer is incorporated in a concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 mg/ml. In certain embodiments of the present invention, sucrose or trehalose are used as stabilizing agents.

[0078] If desired, the formulations also include appropriate amounts of bulking and osmolarity regulating agents. Bulking agents include, for example and without limitation, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In one embodiment, the bulking agent is sucrose. The bulking agent is incorporated in a concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200 mg/ml.

[0079] Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules with the capability of out-competing proteins for interfacial positions. Hydrophobic portions of the surfactant molecules occupy interfacial positions (e.g., air/liquid), while hydrophilic portions of the molecules remain oriented towards the bulk solvent. At sufficient concentrations (typically around the detergent's critical micellar concentration), a surface layer of surfactant molecules serve to prevent protein molecules from adsorbing at the interface. Thereby, surface-induced degradation is minimized. Surfactants contemplated herein include, without limitation, fatty acid esters of sorbitan polyethoxylates, i.e. polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that imparts hydrophobic character to the molecules, C-12 and C-18, respectively. Accordingly, polysorbate-80 is more surface-active and has a lower critical micellar concentration than polysorbate-20.

[0080] In the present formulations, the surfactant is incorporated in a concentration of about 0.01 to about 0.5 g/L. In formulations provided, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g/L (also referred to as % weight/volume). The preferred surfactant is polysorbate-80.

Exemplary Formulations

[0081] The antibody and ADC formulations disclosed herein are suitable for both liquid and lyophilized formulations. That is, the antibody and ADC formulations, e.g., of SGN-CD19A, can be prepared in the disclosed concentrations and stored as a liquid formulation until administered to a patient. Alternatively, a liquid formulation can be prepared, e.g., of SGN-CD19A, in the disclosed concentrations then lyophilized and stored in state, until reconstituted and administered to a patient.

[0082] In certain embodiments, the stable formulation comprises an anti-CD19 antibody at a concentration of between 5 and 25 mg/ml. In other embodiments, the anti-CD19 antibody is included at between 10 and 20 mg/ml. In other embodiments, the anti-CD19 antibody is included at a concentration between 12.5 and 17.5 mg/ml. In other embodiments, the anti-CD19 antibody is included at a concentration between 14 and 16 mg/ml. In other embodiments, the anti-CD19 antibody is included at a concentration of about 15 mg/ml. In other embodiments, the anti-CD19 antibody is included at a concentration of 15 mg/ml.

[0083] In certain embodiments, the stable formulation comprises an anti-CD19 antibody conjugated to a cytotoxic agent at a concentration of between 5 and 25 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to a cytotoxic agent is included at between 10 and 20 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to a cytotoxic agent is included at a concentration between 12.5 and 17.5 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to a cytotoxic agent is included at a concentration between 14 and 16 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to a cytotoxic agent is included at a concentration of about 15 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to a cytotoxic agent is included at a concentration of 15 mg/ml.

[0084] In certain embodiments, the stable formulation comprises an anti-CD19 antibody conjugated to an auristatin at a concentration of between 5 and 25 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to an auristatin is included at between 10 and 20 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to an auristatin is included at a concentration between 12.5 and 17.5 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to an auristatin is included at a concentration between 14 and 16 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to an auristatin is included at a concentration of about 15 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to an auristatin is included at a concentration of 15 mg/ml.

[0085] In certain embodiments, the stable formulation comprises an anti-CD19 antibody conjugated to MMAF at a concentration of between 5 and 25 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to MMAF is included at between 10 and 20 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to MMAF is included at a concentration between 12.5 and 17.5 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to MMAF is included at a concentration between 14 and 16 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to MMAF is included at a concentration of about 15 mg/ml. In other embodiments, the anti-CD19 antibody conjugated to MMAF is included at a concentration of 15 mg/ml.

[0086] In certain embodiments, the stable formulation comprises an anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF at a concentration of between 5 and 25 mg/ml. In other embodiments, the anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF is included at between 10 and 20 mg/ml. In other embodiments, the anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF is included at a concentration between 12.5 and 17.5 mg/ml. In other embodiments, the anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF is included at a concentration between 14 and 16 mg/ml. In other embodiments, the anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF is included at a concentration of about 15 mg/ml. In other embodiments, the anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to MMAF is included at a concentration of 15 mg/ml.

[0087] In certain embodiments, the stable formulation comprises a phosphate buffer with a pH value between 5.0 and 7.0. In other embodiments, the stable formulation comprises a phosphate buffer with a pH value between 5.5 and 6.5. In other embodiments, the stable formulation comprises a phosphate buffer with a pH value between 5.8 and 6.2. In other embodiments, the stable formulation comprises a phosphate buffer with a pH value between 5.9 and 6.1. In other embodiments, the stable formulation comprises a phosphate buffer with a pH value of about 6.0. In other embodiments, the stable formulation comprises a phosphate buffer with a pH value of 6.0

[0088] In certain embodiments, the stable formulation comprises between 0.0% and 1% (W/V) polysorbate 80. In other embodiments, the stable formulation comprises between 0.05% and 0.5% (W/V) polysorbate 80. In other embodiments, the stable formulation comprises between 0.1% and 0.3% (W/V) polysorbate 80. In other embodiments, the stable formulation comprises about 0.2% (W/V) polysorbate 80. In other embodiments, the stable formulation comprises 0.2% (W/V) polysorbate 80.

[0089] In certain embodiments, the stable formulation comprises between 20 and 100 mg/ml sucrose. In other embodiments, the stable formulation comprises between 30 and 90 mg/ml sucrose. In other embodiments, the stable formulation comprises between 40 and 80 mg/ml sucrose. In other embodiments, the stable formulation comprises between 50 and 70 mg/ml sucrose. In other embodiments, the stable formulation comprises between 55 and 65 mg/ml sucrose. In other embodiments, the stable formulation comprises about 60 mg/ml sucrose. In other embodiments, the stable formulation comprises 60 mg/ml sucrose.

[0090] In one embodiment, the stable formulation comprises an anti-CD19 antibody at a concentration of about 15 mg/ml in 10 mM potassium phosphate, at a pH of about 6.0, with about 0.02% w/v polysorbate 80, and about 60 mg/ml sucrose.

[0091] In another embodiment, the stable formulation comprises an anti-CD19 antibody at a concentration of 15 mg/ml in 10 mM potassium phosphate, at pH 6.0, with 0.02% w/v polysorbate 80, and 60 mg/ml sucrose.

[0092] In one embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to a cytotoxic agent at a concentration of about 15 mg/ml in 10 mM potassium phosphate, at a pH of about 6.0, with about 0.02% w/v polysorbate 80, and about 60 mg/ml sucrose.

[0093] In another embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to a cytotoxic agent at a concentration of 15 mg/ml in 10 mM potassium phosphate, at pH 6.0, with 0.02% w/v polysorbate 80, and 60 mg/ml sucrose.

[0094] In one embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to an auristatin at a concentration of about 15 mg/ml in 10 mM potassium phosphate, at a pH of about 6.0, with about 0.02% w/v polysorbate 80, and about 60 mg/ml sucrose.

[0095] In another embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to an auristatin at a concentration of 15 mg/ml in 10 mM potassium phosphate, at pH 6.0, with 0.02% w/v polysorbate 80, and 60 mg/ml sucrose.

[0096] In one embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to mcMMAF at a concentration of about 15 mg/ml in 10 mM potassium phosphate, at a pH of about 6.0, with about 0.02% w/v polysorbate 80, and about 60 mg/ml sucrose.

[0097] In another embodiment, the stable formulation comprises an anti-CD19 antibody conjugated to mcMMAF at a concentration of 15 mg/ml in 10 mM potassium phosphate, at pH 6.0, with 0.02% w/v polysorbate 80, and 60 mg/ml sucrose.

[0098] In one embodiment, the stable formulation comprises an anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to mcMMAF at a concentration of about 15 mg/ml in 10 mM potassium phosphate, at a pH of about 6.0, with about 0.02% w/v polysorbate 80, and about 60 mg/ml sucrose.

[0099] In another embodiment, the stable formulation comprises an anti-CD19 antibody comprising a light chain variable region of SEQ ID NO:1 and a heavy chain variable region of SEQ ID NO:2 that is conjugated to mcMMAF at a concentration of 15 mg/ml in 10 mM potassium phosphate, at pH 6.0, with 0.02% w/v polysorbate 80, and 60 mg/ml sucrose.

Methods of Preparation

[0100] The stable formulation of a CD19 antibody or ADC can be prepared as either a liquid or a lyophilized preparation. As disclosed herein, both the liquid and lyophilized versions of the formulation are stable over time. Once a liquid formulation is made it can then be stored before. Preferably storage of the liquid version will occur at or about 4 C.

[0101] For a lyophilized version of the stable formulation, lyophilization is carried out using techniques common in the art. See, e.g., Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996). A lyophilization cycle is, in one aspect, composed of three steps: freezing, primary drying, and secondary drying. See, e.g., A. P. Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977). In the freezing step, the solution is cooled to initiate ice formation. Furthermore, this step induces the crystallization of the bulking agent. The ice sublimes in the primary drying stage, which is conducted by reducing chamber pressure below the vapor pressure of the ice, using a vacuum and introducing heat to promote sublimation. Finally, adsorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and at an elevated shelf temperature. The process produces a material known as a lyophilized cake. Thereafter the cake can be reconstituted with either sterile water or suitable diluent for injection.

[0102] The standard reconstitution practice for lyophilized material is to add back a volume of pure water or sterile water for injection (WFI) (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration. See, e.g, Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992). Accordingly, methods are provided for preparation of reconstituted stable formulations comprising the step of adding a diluent to a lyophilized CD19 ADC formulation.

[0103] The lyophilized material may be reconstituted as an aqueous solution. A variety of aqueous carriers, e.g., sterile water for injection, or water with appropriate amounts of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions).

[0104] The stable CD19 ADC formulations disclosed herein are administered to patients in need of treatment, e.g., patients with cancer that express extracellular CD19, e.g., non-hodgkin lymphoma or acute lymphoblastic leukemia or patients with an autoimmune disease that responds to treatment with a CD19 ADC. Typically, the CD19 formulations, either liquid or reconstituted lyophilized formulations, are administered intravenously.

[0105] Single or multiple administrations of the compositions are carried out with the dose levels and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dosage depends on the type of disease to be treated, as defined above, the severity and course of the disease, whether drug is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.

[0106] As an additional aspect, the invention includes kits which comprise one or more liquid or lyophilized compositions packaged in a manner which facilitates their use for administration to subjects. In one embodiment, such a kit includes a stable pharmaceutical formulation described herein (e.g., a composition comprising a CD19 ADC, such as SGN-CD19A), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition in practicing the method. In one embodiment, the pharmaceutical formulation is packaged in the container such that the amount of headspace in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of headspace is negligible (i.e., almost none). In one embodiment, the kit contains a first container having a lyophilized stable CD19 ADC formulation, such as SGN-CD19A, and a second container having a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in a unit dosage form. The kit may further include a device suitable for administering the pharmaceutical formulation according to a specific route of administration. Preferably, the kit contains a label that describes use of the pharmaceutical formulations.

[0107] The following examples are not intended to be limiting but only exemplary of specific embodiments of the invention.

EXAMPLES

[0108] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1: Optimization of Antibody Photostability

[0109] Antibody hBU12 was observed to be unstable in the presence of light. Formulation studies indicated that buffer composition could influence the extent of photo-oxidation in hBU12. The following buffers were tested under intense light for their effect on the photostability of hBU12: 10 mM Na phosphate pH 6.5; 10 mM citrate, pH 6.5; and 10 mM histidine, pH 6.5. Each formulation had an hBU12 concentration of 7 mg/mL. The samples were held in a photochamber and exposed to intense light over a period of twenty-four hours. Photo oxidation of hBU12 resulted increase in the oxidized Fab. The data in Table 1 shows change in oxidized Fab for each formulation over time and light exposure. The data indicates that the sodium phosphate formulation showed superior stability compared to citrate and histidine buffers.

TABLE-US-00001 TABLE 1 Effect of buffer on antibody photo stability in intense light. Percent Oxidized Fab Intense Light Exposure Sodium Duration Histidine Phosphate Citrate T = 0 hrs 7.6% 7.3% 8.0% T = 6 hrs 11.3% 9.2% 18.9% T = 18 hrs 21.0% 12.2% 36.2% T = 24 hrs 23.6% 13.2% 43.8% T = 24 hrs Dark Control 7.4% 7.0% 7.8%

[0110] The next study investigated the effect of both intense light and ambient light exposure on another set of formulations. The following buffers were tested for their effect on the photostability of hBU12: 10 mM histidine, pH 6.0; 10 mM potassium phosphate, pH 6.0; 10 mM acetate, pH 6.0; and 10 mM succinate, pH 6.0. The concentration of hBU12 was approximately 4 mg/mL in each formulation.

[0111] Results are shown in Tables 2 and 3. When tested in intense light, hBU12 antibody formulated in a phosphate buffer at pH 6.0 was more photostable than the same antibody formulated in histidine, acetate or succinate buffers. See, e.g., Table 2. Table 3 provides the results in ambient light. Again, hBU12 antibody formulated in a phosphate buffer at pH 6.0 was more photostable than the same antibody formulated in histidine, acetate or succinate buffers.

TABLE-US-00002 TABLE 2 Effect of buffer on antibody photo stability in intense light. Intense light exposure Percent Oxidized Fab duration (days) Histidine K Phosphate Acetate Succinate T = 0 hrs 6.6% 5.1% 6.5% 6.9% T = 12 hrs 10.9% 6.7% 8.1% 10.1% T = 24 hrs 14.9% 7.1% 11.0% 12.5% T = 24 hrs Dark Control 6.5% 4.2% 6.5% 7.1%

TABLE-US-00003 TABLE 3 Effect of buffer on antibody photostability in ambient light. Ambient light exposure Percent Oxidized Fab duration (days) Histidine K Phosphate Acetate Succinate T = 0 d 6.6% 5.1% 6.5% 6.9% T = 3 d 7.9% 4.0% 6.7% 7.9% T = 11 d 9.8% 4.5% 7.6% 8.6% T = 11 d Dark Control 6.8% 3.1% 6.3% 7.0%

[0112] An additional photostability study was conducted for two hBU12 antibody formulations. For this study, the photostability of hBU12 antibody formulated in 10 mM histidine, pH 6.0 was compared to 10 mM K phosphate, pH 6.0 at an hBU12 concentration of 6 mg/mL. The formulations were subjected to intense light and the oxidation results are summarized in Table 4.

[0113] The results shown in Table 4 indicate that hBU12 antibody formulated in a potassium phosphate buffer at pH 6.0 was more photostable than the same antibody formulated in histidine buffer.

TABLE-US-00004 TABLE 4 Photostability of potassium phosphate and histidine formulations in intense light Intense light exposure % Oxidized Fab duration (hours) Histidine K Phosphate 0 8.7% 8.6% 19 h 18.3% 12.5% 19 h Dark Control 9.0% 8.6%

[0114] The hBU12 antibody was conjugated to the auristatin monomethylauristatin F (MMAF) using a maleimidocaproyl (mc) linker. The structure of the mc-MMAF drug linker, methods of making it, and methods of conjugating the drug linker to the hBU12 antibody are disclosed, e.g., in McDonagh et al., WO2009/052431.

[0115] The effect of buffer type and pH on the photostability of the hBU12 mAb was investigated at ambient light exposure. Formulations were prepared in 10 mM histidine buffers at pH 5.0 (H5), pH 6.0 (H6), and pH 7.0 (H7), as well as 10 mM potassium phosphate buffers at pH 6.0 (P6) and pH 7.0 (P7). The photostability of the hBU12 antibody was measured at zero, three and seven days of ambient light exposure. The monoclonal antibody results are shown in FIG. 1A. As expected, the monoclonal antibody was most photostable in phosphate buffer at pH 6.0 (P6).

[0116] Phosphate buffer also stabilized the hBU12 ADC in the presence of light. The evaluation was done with phosphate and histidine buffers, at pH values ranging from 5.0 to 7.0. Formulations were prepared in 10 mM histidine buffers at pH 5.0 (H5), pH 6.0 (H6), and pH 7.0 (H7), and 10 mM potassium phosphate buffers at pH 6.0 (P6) and pH 7.0 (P7) The photostability of the CD19-ADC comprising hBU12 and MMAF was measured at zero and seven days of ambient light exposure. As before, the photostability was assessed by measuring the amount of oxidized Fab or ADC produced under various conditions FIG. 1B shows that the CD19 ADC comprising hBU12 and MMAF was most photostable in phosphate buffer at pH 6.0 (P6).

[0117] The effect of pH on the chemical stability of a CD19 ADC comprising hBU12 and MMAF was determined. FIG. 2 provides the results. The CD19 ADC was formulated at pH 5, 6 and 7 and those formulations were tested over 14 days storage at 40 C. The most significant changes in the molecule were formation of high molecular weight (HMW) species, low molecular weight species (LMW), acidic variants (AV) and basic variants (BV). FIG. 2 shows the percent change from initial for each degradant species following fourteen days at 40 C. FIG. 2 shows that some degradants grew faster at high pH while others grew faster at low pH. Overall, the CD19 ADC comprising hBU12 and MMAF was most stable at pH 6.0.

Example 2: Development of Liquid and Lyophilized Formulations

[0118] The effect of polysorbate 80 on the physical stability of hBU12 formulation was studied. FIGS. 3-6 show that the addition of polysorbate 80 improved the physical stability of hBU12 during freeze/thaw stress. FIGS. 3A and 3B indicate that the size and number of subvisible particles were increased significantly in hBU12 formulation during freeze/thaw in the absence of polysorbate 80. FIGS. 4-6 show that addition of polysorbate 80 at levels of 0.01%, 0.02% and 0.03% significantly reduced the amount of subvisible particles formed under the same conditions of freeze/thaw stress.

[0119] The effect of sugars on stability of a lyophilized formulation of a CD19 ADC comprising hBU12 and MMAF was assessed. FIG. 7 summarizes data on the effect of two sugars, sucrose and trehalose, on the stability of CD19 ADC lyophilized formulations. The data indicates that both sugars impart stability on the CD19 ADC. Higher levels of both sugars impart greater stability and sucrose is more effective than trehalose at the same level.

[0120] Tables 5 and 6 demonstrate the effects of sucrose on aggregate and fragment formation in the lyophilized formulation of a CD19 ADC comprising hBU12 and MMAF over a two week period under stressed conditions, i.e., at 54 C. Lowest levels of aggregates and fragments (greatest stability) were seen after two weeks in the presence of 60 mg/ml (6% w/v) sucrose.

TABLE-US-00005 TABLE 5 Effect of sucrose on lyophilization stability - measurement of aggregates Aggregate Aggregate Aggregate Aggregate Aggregate Post-Lyo, Post-Lyo, Post-Lyo, Pre-Lyo Post-Lyo 3 days @ 1 week @ 2 weeks @ Sample initial initial 54 C. 54 C. 54 C. PDN-290-02-68, 1.3 1.3 1.6 1.7 2.1 30 mg/mL Sucrose PDN-290-02-68, 1.3 1.3 1.5 1.5 1.7 45 mg/mL Sucrose PDN-290-02-68, 1.3 1.3 1.4 1.5 1.6 60 mg/mL Sucrose

TABLE-US-00006 TABLE 6 Effect of sucrose on lyophilization stability - measurement of fragments Fragment Fragment Fragment Fragment Fragment Post-Lyo, Post-Lyo, Post-Lyo, Pre-Lyo Post-Lyo 3 days @ 1 week @ 2 weeks @ Sample initial initial 54 C. 54 C. 54 C. PDN-290-02-68, 0.8 0.8 0.9 1.0 1.0 30 mg/mL Sucrose PDN-290-02-68, 0.8 0.8 0.9 0.9 0.9 45 mg/mL Sucrose PDN-290-02-68, 0.8 0.8 0.9 0.9 0.9 60 mg/mL Sucrose

[0121] Based on the results of development studies, a formulated solution of CD19A ADC at 15 mg/mL in 10 mM phosphate, 6% w/v sucrose, 0.02% w/v polysorbate 80 was selected for further development. The stability of this formulation has been examined in both the liquid and lyophilized state. For the liquid drug product, the formulated solution is filled directly into vials and stored as a liquid for long-term storage. For the lyophilized drug product, the formulated solution is filled into vials and lyophilized to produce a solid product for long-term storage.

[0122] The long-term stability of the liquid drug product is excellent as shown in Table 7. Most product quality attributes of the liquid drug product do not change significantly following 18 or 36 months of storage at 5 C. In particular, the biological assays and drug distribution on the ADC do not change. Attributes that do change slightly are high molecular weight (HMW) species, low molecular weight (LMW) species and acidic variants (AV). The changes in these attributes from initial following 18 months storage at 5 C. for two liquid drug product lots are summarized in Table 7, as are the results at 36 months for drug lot DEVGLY-1.

TABLE-US-00007 TABLE 7 Change in HMW, LMW and AV of Liquid Drug Product Following 18 Months Storage at 5 C. Attribute Change DEVGLY-1 DEVGLY-2 % HMW +0.4% 18 M/5 C. +0.5% 18 M/5 C. +0.6% 36 M/5 C. % LMW +0.8% 18 M/5 C. +1.0% 18 M/5 C. +1.3% 36 M/5 C. % AV +1.6% 18 M/5 C. +2.2% 18 M/5 C. +6.4% 36 M/5 C.

[0123] The long-term stability of the lyophilized drug product is excellent as shown in Table 8. Most product quality attributes of the lyophilized drug product do not change significantly following 18, 30 or 36 months of storage at 25 C. and 12 months of storage at 40 C. In particular, the biological assays and drug product distribution on the ADC do not change. Stress storage at 40 C. shows slight change in some product attributes including high molecular weight (HMW) species, low molecular weight (LMW) species and basic variants (BV). At 25 C. long-term storage there is a slight increase in HMW and no other changes. The changes in these attributes from initial following 18, 30 or 36 months storage at 25 C. and 12 months of storage at 40 C. are summarized in Table 8.

TABLE-US-00008 TABLE 8 Change in HMW, LMW and BV of Lyophilized Drug Product Following 18, 30 or 36 Months Storage at 25 C. and 12 Months Storage at 40 C. Attribute Change DEVSHW-1 DEVSHW-2 SHW001 % HMW NC 18 M/25 C. +0.3% 18 M/25 C. +0.3% 18 M/25 C. +0.3% 36 M/25 C. +0.5% 36 M/25 C. +0.4% 30 M/25 C. +0.6% 12 M/40 C. +0.6% 12 M/40 C. % LMW NC 18 M/25 C. NC 18 M/25 C. NC 18 M/25 C. +0.1% 36 M/25 C. +0.1% 36 M/25 C. +0.1% 30 M/25 C. +0.4% 12 M/40 C. +0.3% 12 M/40 C. % BV NC 18 M/25 C. NC 18 M/25 C. NC 18 M/25 C. +1.1% 36 M/25 C. +1.2% 36 M/25 C. +1.5% 30 M/25 C. +2.4% 12 M/40 C. +3.1% 12 M/40 C.

[0124] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

INFORMAL SEQUENCE LISTING

[0125]

TABLE-US-00009 hBU12lightchainvariableregion SEQIDNO:1 eivltqspatlslspgeratlscsasssvsymhwyqqkpgqaprlliy dtsklasgiparfsgsgsgtdftltisslepedvavyycfqgsvypft fgqgtkleikr hBU12heavychainvariableregion SEQIDNO:2 qvqlqesgpglvkpsqtlsltctvsggsistsgmgvgwirqhpgkgle wighiwwdddkrynpalksrvtisvdtsknqfslklssvtaadtavyy carmelwsyyfdywgqgtlvtvss