Stabilized formulation containing anti-MUC16 x anti-CD3 bispecific antibody

Abstract

The present invention provides a stable liquid pharmaceutical formulation comprising a human bispecific antibody that specifically binds human MUC16 and human CD3. In certain embodiments, the formulation contains a buffer, a surfactant, and a sugar in addition to the bispecific antibody. The pharmaceutical formulation of the present invention exhibits a significant degree of antibody stability upon stress and storage.

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference a sequence listing submitted in computer readable form as file 10820WO01-Sequence.txt, created on April 1, 2022, and containing 25,436 bytes.

[0002] The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations comprising a human bispecific antibody that specifically binds to human MUC16 and human CD3. [Background technology]

[0003] Therapeutic macromolecules (e.g., antibodies) must be formulated in a manner that not only renders the molecule suitable for administration to a patient, but also maintains its stability during storage and subsequent use. For example, therapeutic antibodies in liquid solutions are prone to degradation, aggregation, and / or undesirable chemical modifications unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation, but also on the amounts and ratios of the excipients relative to each other. Furthermore, other considerations besides stability must be taken into account when preparing liquid antibody formulations. Examples of such additional considerations include the concentration of antibody that can be accommodated by a given formulation, and the visual quality or appeal of the formulation. Thus, great care must be taken when formulating therapeutic antibodies to obtain a formulation that remains stable, contains an appropriate concentration of antibody, and possesses other properties that allow the formulation to be conveniently administered to a patient.

[0004] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a single transmembrane domain, highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer. CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation.

[0005] A bispecific antibody against human MUC16 and human CD3 is an example of a therapeutically relevant macromolecule that requires appropriate formulation. Such antibodies are clinically useful, for example, for the treatment of cancer (e.g., MUC16-expressing cancers, ovarian cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer).

[0006] While anti-MUC16 x anti-CD3 bispecific antibodies are known in the art (see, e.g., WO2018 / 067331), there remains a need for pharmaceutical formulations comprising anti-MUC16 x anti-CD3 bispecific antibodies that are sufficiently stable and suitable for administration to patients. Summary of the Invention

[0007] Provided are stable liquid pharmaceutical formulations comprising a bispecific anti-MUC16 x anti-CD3 antibody and one or more excipients, as well as kits, unit dosage forms, and containers comprising such formulations and uses thereof.

[0008] In one aspect, the invention provides a bispecific antibody comprising (a) a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) comprised in a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR); and the second antigen-binding domain comprises three heavy chain CDRs (A2-HCDR1, A2-HCDR2, and A2-HCDR3) comprised in the heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in the light chain variable region (LCVR). and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprising A1-HCDR1, A1-HCDR2, and A1-HCDR3, respectively, wherein A1-HCDR1, A1-HCDR2, and A1-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, A2-HCDR1, A2-HCDR2, and A2-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (b) a buffer comprising sodium acetate; (c) an organic co-solvent comprising a polysorbate; and (d) a stabilizer comprising a sugar, wherein the formulation has a pH of 5.0±0.5.

[0009] In some cases, the antibody concentration is 1 mg / ml ± 0.1 mg / ml to 200 mg / ml ± 20 mg / ml. In some cases, the antibody concentration is 5 mg / ml ± 0.5 mg / ml to 50 mg / ml ± 5 mg / ml. In some cases, the antibody concentration is 5 mg / ml ± 0.5 mg / ml. In some cases, the antibody concentration is 50 mg / ml ± 5 mg / ml. In some cases, the antibody concentration is 150 mg / ml ± 15 mg / ml.

[0010] In some cases, the acetate buffer concentration is 10 mM ± 1 mM to 50 mM ± 5 mM. In some cases, the acetate buffer concentration is 25 mM ± 2.5 mM to 35 mM ± 3.5 mM. In some cases, the acetate buffer concentration is 30 mM ± 3 mM.

[0011] In some embodiments, the polysorbate concentration is 0.01%±0.005% to 0.5%±0.05% w / v. In some embodiments, the polysorbate concentration is 0.1%±0.05% to 0.3%±0.03% w / v. In some embodiments, the polysorbate concentration is 0.2%±0.02% w / v. In some embodiments, the polysorbate concentration is 0.05%±0.01% w / v. In some embodiments, the polysorbate is polysorbate 20.

[0012] In some embodiments, the sugar is sucrose. Optionally, the sucrose concentration is 5%±1% to 20%±4% w / v. Optionally, the sucrose concentration is 7%±0.5% to 12%±0.5% w / v. Optionally, the sucrose concentration is 10%±1% w / v. Optionally, the sucrose concentration is 7%±0.7% w / v. Optionally, the sucrose concentration is 8%±0.8% w / v.

[0013] In some embodiments, the pharmaceutical formulation comprises (a) 5 mg / ml ± 0.5 mg / ml antibody, (b) 25 mM ± 2 mM to 35 mM ± 2 mM acetate buffer, (c) 0.1% ± 0.05% to 0.3% ± 0.05% w / v polysorbate, and (d) 5% ± 1% to 15% ± 3% w / v sucrose, all at pH 5.0 ± 0.5.

[0014] In some embodiments, the pharmaceutical formulation comprises (a) 5 mg / ml ± 0.5 mg / ml of antibody, (b) 30 mM ± 1 mM acetate buffer, (c) 0.2% ± 0.02% w / v polysorbate, and (d) 10% ± 1% w / v sucrose at pH 5.0 ± 0.3.

[0015] In some embodiments, the pharmaceutical formulation comprises: (a) 50 mg / ml ± 5 mg / ml antibody, (b) 25 mM ± 2 mM to 35 mM ± 2 mM acetate buffer, (c) 0.1% ± 0.05% to 0.3% ± 0.05% w / v polysorbate, and (d) 5% ± 1% to 15% ± 3% w / v sucrose, all at pH 5.0 ± 0.5.

[0016] In some embodiments, the pharmaceutical formulation comprises (a) 50 mg / ml ± 0.5 mg / ml of antibody, (b) 30 mM ± 1 mM acetate buffer, (c) 0.2% ± 0.02% w / v polysorbate, and (d) 10% ± 1% w / v sucrose at pH 5.0 ± 0.3.

[0017] In any of these embodiments, the polysorbate may be polysorbate 20.

[0018] In any of the various embodiments discussed above, the formulation contains 2.5% or less high molecular weight (HMW) species after 12 months or 24 months storage at 5° C. as determined by size exclusion ultra performance liquid chromatography (SE-UPLC). In some cases, the formulation contains 3.5% or less high molecular weight (HMW) species after 6 months storage at 25° C. and 60% relative humidity as determined by SE-UPLC. In some cases, the formulation contains 1.5% or less high molecular weight (HMW) species after 12 months storage at −30° C. or 2.0% or less HMW species after 24 months storage at −30° C. as determined by SE-UPLC. In some cases, the formulation contains 1.5% or less high molecular weight (HMW) species after 12 months storage at −80° C. or 2.0% or less HMW species after 24 months storage at −30° C. as determined by SE-UPLC.

[0019] In one aspect, the invention provides (a) a bispecific antibody at a concentration of 1 mg / ml to 30 mg / ml, the bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) comprised in a heavy chain variable region (HCVR) and three light chains (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR), and the second antigen-binding domain comprising three heavy chain CDRs (A2-HCDR1, A2-HCDR2, and A2-HCDR3) comprised in the heavy chain variable region (HCVR) and three light chains (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR). and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in A1-HCDR1, A1-HCDR2, and A1-HCDR3, respectively, wherein A1-HCDR1, A1-HCDR2, and A1-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, A2-HCDR1, A2-HCDR2, and A2-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (b) a buffer comprising histidine; (c) an organic co-solvent comprising a polysorbate; and (d) a stabilizer comprising a sugar, wherein the formulation has a pH of 6.0±0.5.

[0020] In some embodiments, the antibody concentration is 2 mg / ml ± 0.5 mg / ml. In some embodiments, the antibody concentration is 20 mg / ml ± 2 mg / ml. In some embodiments, the histidine buffer concentration is 5 mM ± 1 mM to 15 mM ± 1 mM. In some embodiments, the histidine buffer concentration is 10 mM ± 1 mM. In some embodiments, the polysorbate concentration is 0.01% to 0.1% w / v. In some embodiments, the polysorbate concentration is 0.05% ± 0.01% w / v. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose concentration is 8% ± 0.5% to 12% ± 0.5% w / v. In some embodiments, the sucrose concentration is 10% ± 1% w / v.

[0021] In some of the embodiments of this aspect of the invention, (a) at least 95% of the antibody retains the native conformation after 12 months, 18 months, 24 months, or 36 months of storage at 5° C.; (b) at least 95% of the antibody retains the native conformation after 6 months of storage at 25° C. and 60% relative humidity; (c) at least 95% of the antibody retains the native conformation after 3 months of storage at 37° C.; (d) the formulation contains 1% or less high molecular weight (HMW) species after 12 months, 18 months, 24 months, or 36 months of storage at 5° C.; (e) the formulation contains 1% or less HMW species after 6 months of storage at 25° C. and 60% relative humidity; or (f) the formulation contains 1% or less HMW species after 3 months of storage at 37° C., as determined by SE-UPLC.

[0022] In one aspect, the invention provides (a) a bispecific antibody at a concentration of 100 mg / ml to 200 mg / ml, the bispecific antibody comprising a first antigen-binding domain that specifically binds to human MUC16 and a second antigen-binding domain that specifically binds to human CD3, the first antigen-binding domain comprising three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) comprised in a heavy chain variable region (HCVR) and three light chains (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR), and the second antigen-binding domain comprising three heavy chain CDRs (A2-HCDR1, A2-HCDR2, and A2-HCDR3) comprised in the heavy chain variable region (HCVR). and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in the light chain variable region (LCVR), wherein A1-HCDR1, A1-HCDR2, and A1-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, A2-HCDR1, A2-HCDR2, and A2-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (b) a buffer comprising an acetate salt; (c) a stabilizer comprising a sugar; and (d) a surfactant comprising a polysorbate, wherein the formulation has a pH of 5.0±0.5.

[0023] In some embodiments, the antibody concentration is 125 mg / ml to 175 mg / ml. In some embodiments, the antibody concentration is 150 mg / ml ± 10 mg / ml. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose concentration is 4% to 12% w / v. In some embodiments, the sucrose concentration is 8% w / v ± 1% w / v. In some embodiments, the acetate buffer concentration is 25 mM to 35 mM. In some embodiments, the acetate buffer concentration is 30 mM ± 1 mM. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate 20 concentration is 0.01% w / v to 0.1% w / v. In some embodiments, the polysorbate 20 concentration is 0.05% w / v ± 0.01% w / v.

[0024] In some of the embodiments of this aspect of the invention, (a) the formulation contains 2.5% or less high molecular weight (HMW) species after 12 or 24 months of storage at -30°C or -80°C, (b) the formulation contains 4% or less HMW species after 6 months of storage at 5°C, or (c) the formulation contains 6% or less HMW species after 6 months of storage at 25°C and 60% relative humidity, as determined by SE-UPLC.

[0025] In any of the various embodiments described above or discussed herein, the formulation contains 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, or 35% or less of a glycosylated species variant, where the glycosylated species variant comprises a glycosylation at residue 98 of SEQ ID NO:1 or SEQ ID NO:4, or at residue 2 of SEQ ID NO:9.

[0026] In any of the various embodiments described above or discussed herein, the first antigen-binding domain comprises an HCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:4 and an LCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:6, and the second antigen-binding domain comprises an HCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:5 and an LCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:6.

[0027] In any of the various embodiments described above or discussed herein, the first antigen-binding domain comprises an HCVR having at least 95% identity to the amino acid sequence of SEQ ID NO:4 and an LCVR having at least 95% identity to the amino acid sequence of SEQ ID NO:6, and the second antigen-binding domain comprises an HCVR having at least 95% identity to the amino acid sequence of SEQ ID NO:5 and an LCVR having at least 95% identity to the amino acid sequence of SEQ ID NO:6.

[0028] In any of the various embodiments described above or discussed herein, the first antigen-binding domain comprises an HCVR having at least 99% identity to the amino acid sequence of SEQ ID NO:4 and an LCVR having at least 99% identity to the amino acid sequence of SEQ ID NO:6, and the second antigen-binding domain comprises an HCVR having at least 99% identity to the amino acid sequence of SEQ ID NO:5 and an LCVR having at least 99% identity to the amino acid sequence of SEQ ID NO:6.

[0029] In any of the various embodiments described above or discussed herein, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6.

[0030] In one aspect, the invention provides a stable pharmaceutical formulation comprising: (a) a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, at 5 mg / ml ± 0.5 mg / ml, wherein the first antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 4 and a LCVR that comprises an amino acid of SEQ ID NO: 6, and the second antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 5 and a LCVR that comprises an amino acid of SEQ ID NO: 6; (b) 30 mM ± 1 mM sodium acetate buffer at pH 5.0 ± 0.2; (c) 0.2% ± 0.02% w / v polysorbate 20; and (d) 10% ± 1% w / v sucrose.

[0031] In one aspect, the invention provides a stable pharmaceutical formulation comprising: (a) a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, at 50 mg / ml ± 5 mg / ml, wherein the first antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 4 and a LCVR that comprises an amino acid of SEQ ID NO: 6, and the second antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 5 and a LCVR that comprises an amino acid of SEQ ID NO: 6; (b) a 30 mM ± 1 mM sodium acetate buffer at pH 5.0 ± 0.2; (c) 0.2% ± 0.02% w / v polysorbate 20; and (d) 10% ± 1% w / v sucrose.

[0032] In one aspect, the invention provides a stable pharmaceutical formulation comprising: (a) a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, at 150 mg / ml ± 15 mg / ml, wherein the first antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 4 and a LCVR that comprises an amino acid of SEQ ID NO: 6, and the second antigen binding domain comprises a HCVR that comprises an amino acid of SEQ ID NO: 5 and a LCVR that comprises an amino acid of SEQ ID NO: 6; (b) a 30 mM ± 1 mM sodium acetate buffer at pH 5.0 ± 0.2; (c) 0.05% ± 0.01% w / v polysorbate 20; and (d) 8% ± 1% w / v sucrose.

[0033] In any of the various embodiments described above or discussed herein, the antibody comprises a human IgG heavy chain constant region attached to the HCVR of each of the first and second antigen binding domains, respectively. Optionally, the heavy chain constant region is of isotype IgG1. Optionally, the heavy chain constant region is of isotype IgG4.

[0034] In some embodiments, the heavy chain constant region attached to the HCVR of the first antigen-binding domain or the heavy chain constant region attached to the HCVR of the second antigen-binding domain, but not both, comprises an amino acid modification that reduces Protein A binding relative to a heavy chain of the same isotype lacking the modification. Optionally, the modification comprises a H435R substitution (EU numbering) in a heavy chain of isotype IgG1 or IgG4. Optionally, the modification comprises a H435R substitution and a Y436F substitution (EU numbering) in a heavy chain of isotype IgG1 or IgG4.

[0035] In any of the various embodiments described above or discussed herein, the antibody comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some aspects, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 16, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17. In some aspects, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19.

[0036] In any of the various embodiments described above or discussed herein, the antibody comprises a first heavy chain comprising an HCVR of a first antigen binding domain and a second heavy chain comprising an HCVR of a second antigen binding domain, wherein the first heavy chain comprises residues 1-442 of the amino acid sequence of SEQ ID NO: 1 and the second heavy chain comprises residues 1-449 of the amino acid sequence of SEQ ID NO: 2. In some embodiments, the antibody comprises a common light chain comprising an LCVR of the first and second antigen binding domains, wherein the common light chain comprises the amino acid sequence of SEQ ID NO: 3.

[0037] In any of the various embodiments discussed above or herein, the pharmaceutical formulation may be considered stable by measuring the rate of change of "glycated species" that is (i) 1.5% or less after 6 months of storage at 5°C, (ii) 3% or less after 12 months of storage at 5°C, (iii) 1.5% or less after 12 months, 18 months, or 24 months of storage at -30°C, or 1% or less after 12 months, 18 months, or 24 months of storage at -80°C, as determined by cation exchange ultra performance liquid chromatography (CEX-UPLC) and / or by liquid chromatography mass spectrometry (LC-MS).

[0038] In one aspect, the invention provides a pharmaceutical composition, the composition comprising a pharmaceutical formulation as described above or discussed herein, the composition being contained in a container.

[0039] In some embodiments, the container is a vial. Optionally, the vial is a 2 ml, 5 ml, or 10 ml type 1 clear glass vial. In some embodiments, the container is a syringe. Optionally, the syringe is low tungsten glass. In some embodiments, the container is a pre-filled syringe. In some embodiments, the pharmaceutical composition is contained in an autoinjector.

[0040] In one aspect, the invention provides a kit comprising: (i) a container containing a composition comprising a pharmaceutical formulation as described above or discussed herein, and instructions for use of the composition.

[0041] In some embodiments, the container is a glass vial. In some embodiments, the container is a pre-filled syringe. In some embodiments, the container is an autoinjector.

[0042] In some embodiments, the instructions describe subcutaneous administration of the composition. In some embodiments, the instructions describe intravenous administration of the composition.

[0043] In one aspect, the invention provides a unit dosage form comprising a pharmaceutical formulation as described above or discussed herein, wherein the antibody is present in an amount of 0.1 mg to 500 mg. In some cases, the antibody is present in an amount of 5±1 mg to 50±5 mg. In some cases, the antibody is present in an amount of 10±1 mg to 200±20 mg. In some embodiments, the antibody is present in an amount of 4 mg, 5 mg, 10 mg, 12.5 mg, 40 mg, 50 mg, 150 mg, or 180 mg.

[0044] In some embodiments, the unit dosage form is a glass vial, a prefilled syringe, or an autoinjector.

[0045] In one aspect, the invention provides a container comprising a composition comprising a pharmaceutical formulation as described above or discussed herein. In various embodiments, the container is a glass vial, a pre-filled syringe, or an autoinjector.

[0046] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another associated value described above or discussed herein to recite a range with values ​​representing the upper and lower limits of the range, and such ranges and all values ​​within such ranges are encompassed within the scope of the present disclosure. Each of the values ​​described above or discussed herein may be expressed with a 1%, 5%, 10%, or 20% variation. For example, a concentration of 10 mM may be expressed as 10 mM ± 0.1 mM (1% variation), 10 mM ± 0.5 mM (5% variation), 10 mM ± 1 mM (10% variation), or 10 mM ± 2 mM (20% variation).

[0047] Other embodiments will become apparent from consideration of the following detailed description. [Brief description of the drawings]

[0048] [Figure 1]Figure 1 illustrates the relationship between the relative potency of mAb1 and the level of glycosylation of HCDR3-Lys98. Glycosylation levels were generated by purification of glycosylated and non-glycosylated mAb1 by preparative cation exchange chromatography and mixing both species in different ratios. The potency data shows that potency is dependent on the level of glycosylation. [Figure 2A] 2A-2D illustrate the effect of pH on the levels of glycosylated and high molecular weight (HMW) species of mAb1 formulated in histidine buffer. Formulations contained 2 mg / ml mAb1 in 10 mM histidine, 10% w / v sucrose, and 0.05% w / v polysorbate 20 with different pH values ​​and were incubated at 5° C. for up to 36 months or at 25° C. for up to 2 months. Glycation levels were monitored by cation exchange chromatography (CEX-UPLC) at 5° C. (FIG. 2A) or 25° C. (FIG. 2B) and HMW levels were monitored by size exclusion chromatography (SE-UPLC) at 5° C. (FIG. 2C) or 25° C. (FIG. 2D). [Figure 2B] 2A-2D illustrate the effect of pH on the levels of glycosylated and high molecular weight (HMW) species of mAb1 formulated in histidine buffer. Formulations contained 2 mg / ml mAb1 in 10 mM histidine, 10% w / v sucrose, and 0.05% w / v polysorbate 20 with different pH values ​​and were incubated at 5° C. for up to 36 months or at 25° C. for up to 2 months. Glycation levels were monitored by cation exchange chromatography (CEX-UPLC) at 5° C. (FIG. 2A) or 25° C. (FIG. 2B) and HMW levels were monitored by size exclusion chromatography (SE-UPLC) at 5° C. (FIG. 2C) or 25° C. (FIG. 2D). [Figure 2C]2A-2D illustrate the effect of pH on the levels of glycosylated and high molecular weight (HMW) species of mAb1 formulated in histidine buffer. Formulations contained 2 mg / ml mAb1 in 10 mM histidine, 10% w / v sucrose, and 0.05% w / v polysorbate 20 with different pH values ​​and were incubated at 5° C. for up to 36 months or at 25° C. for up to 2 months. Glycation levels were monitored by cation exchange chromatography (CEX-UPLC) at 5° C. (FIG. 2A) or 25° C. (FIG. 2B) and HMW levels were monitored by size exclusion chromatography (SE-UPLC) at 5° C. (FIG. 2C) or 25° C. (FIG. 2D). [Figure 2D] 2A-2D illustrate the effect of pH on the levels of glycosylated and high molecular weight (HMW) species of mAb1 formulated in histidine buffer. Formulations contained 2 mg / ml mAb1 in 10 mM histidine, 10% w / v sucrose, and 0.05% w / v polysorbate 20 with different pH values ​​and were incubated at 5° C. for up to 36 months or at 25° C. for up to 2 months. Glycation levels were monitored by cation exchange chromatography (CEX-UPLC) at 5° C. (FIG. 2A) or 25° C. (FIG. 2B) and HMW levels were monitored by size exclusion chromatography (SE-UPLC) at 5° C. (FIG. 2C) or 25° C. (FIG. 2D). [Figure 3A] FIG. 3 illustrates the effect of pH on the levels of glycated and HMW species of mAb1 formulated in acetate buffer. Formulations contained 50 mg / ml mAb1 in 10 mM acetate and 5% w / v sucrose with different pH values ​​and were incubated at 40° C. for 28 days. Glycation levels were monitored by CEX-UPLC (FIG. 3A) and HMW levels were monitored by SE-UPLC (FIG. 3B). [Figure 3B] FIG. 3 illustrates the effect of pH on the levels of glycated and HMW species of mAb1 formulated in acetate buffer. Formulations contained 50 mg / ml mAb1 in 10 mM acetate and 5% w / v sucrose with different pH values ​​and were incubated at 40° C. for 28 days. Glycation levels were monitored by CEX-UPLC (FIG. 3A) and HMW levels were monitored by SE-UPLC (FIG. 3B). [Figure 4] 1 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on the viscosity of mAb1 formulations. The plots represent statistical modeling of the experimental data. [Diagram 5] 1 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on the osmolality of mAb1 formulations. The plots represent statistical modeling of the experimental data. [Figure 6] 1 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on the stability of mAb1 formulations. The plots represent statistical modeling of the experimental data. [Figure 7A] FIG. 1 illustrates the stability of two mAb1 formulations for subcutaneous administration. Both formulations show comparable stability when incubated under the test conditions of 40° C. / 75% relative humidity (RH) for 3 months, 25° C. / 60% RH for 6 months, and 2-8° C. for 6 months. As shown in the figure, F1 contains 150 mg / ml mAb1, 30 mM acetate at pH 5.0, 8% w / v sucrose, and 0.05% w / v polysorbate 80, and F2 contains 150 mg / ml mAb1, 30 mM acetate at pH 5.0, 7% w / v sucrose, 50 mM arginine, and 0.05% w / v polysorbate 80. [Figure 7B] FIG. 1 illustrates the stability of two mAb1 formulations for subcutaneous administration. Both formulations show comparable stability when incubated under the test conditions of 40° C. / 75% relative humidity (RH) for 3 months, 25° C. / 60% RH for 6 months, and 2-8° C. for 6 months. As shown in the figure, F1 contains 150 mg / ml mAb1, 30 mM acetate at pH 5.0, 8% w / v sucrose, and 0.05% w / v polysorbate 80, and F2 contains 150 mg / ml mAb1, 30 mM acetate at pH 5.0, 7% w / v sucrose, 50 mM arginine, and 0.05% w / v polysorbate 80. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Before describing the invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific recited numerical value or range of values ​​means that the value may vary from the recited value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0051] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0052] Pharmaceutical preparations As used herein, the term "pharmaceutical formulation" refers to a combination of at least one active ingredient (e.g., a bispecific anti-MUC16 x anti-CD3 antibody capable of exerting a biological effect in a human or non-human animal) and at least one inactive ingredient that, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term "formulation" as used herein means "pharmaceutical formulation" unless otherwise indicated. The present invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to certain embodiments of the present invention, the therapeutic polypeptide is a bispecific antibody that specifically binds to human MUC16 and human CD3, or an antigen-binding fragment thereof. More specifically, the present invention includes pharmaceutical formulations comprising, inter alia, (i) a human bispecific antibody that specifically binds to human MUC16 and human CD3, (ii) a buffer comprising an acetate, (iii) an organic co-solvent comprising a polysorbate, and (iv) a stabilizer comprising a sugar. Additional ingredients may be included in the formulations of the present invention, provided that such ingredients do not significantly interfere with the stability of the formulation. Specific exemplary components and formulations included within the present invention are described in detail below.

[0053] The pharmaceutical formulation of the present invention may be a fluid formulation in certain embodiments. As used herein, the term "fluid formulation" refers to a mixture of at least two components that exists in a fluid state primarily at about 2°C to about 45°C. Fluid formulations include, among others, liquid formulations. Fluid formulations may be low, medium, or high viscosity, depending on the particular components.

[0054] A bispecific antibody that specifically binds human MUC16 and human CD3 The pharmaceutical formulations of the invention can comprise a human bispecific antibody, or antigen-binding fragment thereof, that specifically binds to human MUC16 and human CD3.

[0055] The term "antibody" as used herein, including "bispecific antibodies", generally refers to immunoglobulin molecules comprising four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM), although immunoglobulin molecules consisting only of heavy chains (i.e., lacking light chains) are also included within the definition of the term "antibody". Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH), and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL), and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with relatively conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0056] In certain embodiments of the invention, the anti-MUC16×anti-CD3 bispecific antibodies of the invention are human antibodies. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may, for example, include amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In various embodiments, the anti-MUC16×anti-CD3 bispecific antibodies are human IgG antibodies. In various embodiments, the anti-MUC16×anti-CD3 bispecific antibodies are human antibodies of isotype IgG1, IgG2, IgG3, or IgG4, or mixed isotypes. In some embodiments, the anti-MUC16×anti-CD3 bispecific antibody is a human IgG1 antibody (i.e., the antibody comprises a human IgG1 heavy chain constant region attached to the HCVR of each of the first and second antigen binding domains, respectively). In some embodiments, the anti-MUC16×anti-CD3 bispecific antibody is a human IgG4 antibody (i.e., the antibody comprises a human IgG4 heavy chain constant region attached to the HCVR of each of the first and second antigen binding domains, respectively). In any of the embodiments described above or discussed herein, the anti-MUC16×anti-CD3 bispecific antibody may comprise a human kappa light chain. In any of the embodiments described above or discussed herein, the anti-MUC16×anti-CD3 bispecific antibody may comprise a human lambda light chain.

[0057] In any embodiment, the bispecific antibody may comprise a modification in one or both heavy chains to facilitate purification of the bispecific antibody (i.e., heterodimer) from homodimeric impurities. In some embodiments, the bispecific antibody comprises first and second heavy chains (i.e., a heavy chain of an anti-MUC16 binding arm and a heavy chain of an anti-CD3 binding arm) that are identical (e.g., both are isotype IgG1 or IgG4) except for a modification in the CH3 domain of one or the other heavy chain that reduces binding of the bispecific antibody to Protein A compared to an antibody lacking the modification. In some embodiments, the CH3 domain of the first heavy chain (e.g., of the anti-MUC16 binding arm) binds Protein A and the CH3 domain of the second heavy chain (e.g., of the anti-CD3 binding arm) comprises a mutation that reduces or abolishes Protein A binding. In some embodiments, the mutation is an H435R modification (according to EU numbering; H95R according to IMGT exon numbering). In some cases, the mutations are H435R modifications (according to EU numbering; IMGT exon numbering H95R) and Y436F modifications (according to EU numbering; Y96F by IMGT). Further modifications that may be found in the second CH3 domain include: D356E, L358M, N384S, K392N, V397M, and V422I by EU for IgG1 CH3 domains (D16E, L18M, N44S, K52N, V57M, and V82I by IMGT), and Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU (Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I by IMGT) for IgG4 CH3 domains.

[0058] In any embodiment, the bispecific antibody may comprise a chimeric hinge. The term "chimeric hinge" is intended to include a chimeric protein comprising a first amino acid sequence derived from the hinge region of one Ig molecule and a second amino acid sequence derived from the hinge region of an Ig molecule of a different class or subclass. For example, a chimeric hinge, in embodiments, comprises a first amino acid sequence, or "upper hinge" sequence, derived from a human IgG1 hinge region or a human IgG4 hinge region, and a second amino acid sequence, or "lower hinge" sequence, derived from a human IgG2 hinge region. In certain embodiments, the first or "upper hinge" sequence comprises amino acid residues 216-227 according to EU numbering. In some embodiments, the second or "lower hinge" sequence comprises amino acid residues 228-236 according to EU numbering.

[0059] The antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibodies" as used herein is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using recombinant expression vectors transduced into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means that involve splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of animals transgenic for human Ig sequences, in vivo somatic mutagenesis) to thereby determine the V and VL of the recombinant antibody. H Area and V L The amino acid sequence of the region is H Sequence and V LThese are sequences that, while derived from and related to a sequence, may not naturally exist within the human antibody germline repertoire in vivo.

[0060] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to human MUC16 or human CD3.

[0061] An "isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds human MUC16 and human CD3 is substantially free of antibodies that specifically bind to antigens other than human MUC16 and human CD3).

[0062] The term "specifically binds" or equivalents means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6 The specific binding of an antibody to human MUC16 or human CD3 may be characterized by a dissociation constant of M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds human MUC16 and human CD3 may have cross-reactivity to other antigens, such as MUC16 or CD3 molecules from other species (orthologs). In the context of the present invention, a multispecific (e.g., bispecific) antibody that binds human MUC16 and human CD3 and one or more additional antigens is considered to "specifically bind" human MUC16 and human CD3. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0063] Exemplary anti-MUC16 x anti-CD3 bispecific antibodies that may be included in the pharmaceutical formulations of the invention are described in WO2018 / 067331, the disclosure of which is incorporated by reference in its entirety.

[0064] According to certain embodiments of the invention, the anti-MUC16xanti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises heavy chain complementarity determining regions (CDRs) A1-HCDR1, A1-HCDR2, and A1-HCDR3 comprising the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the second antigen-binding domain comprises heavy chain CDRs, A2-HCDR1, A2-HCDR2, and A2-HCDR3 comprising the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively. According to certain embodiments of the invention, the anti-MUC16xanti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a common (for both the first and second antigen-binding domains) light chain complementarity determining region LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively.

[0065] In certain embodiments, the anti-MUC16x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 4 and the second antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the anti-MUC16x anti-CD3 bispecific antibody, or antigen-binding fragment thereof, comprises a first antigen-binding domain comprising a HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 4 / 6, and a second antigen-binding domain comprising a HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO: 5 / 6. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair described above and a human IgG1 heavy chain constant region. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair described above and a human IgG4 heavy chain constant region. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair described above and a human IgG heavy chain constant region. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody comprises the HCVR / LCVR sequence pair described above and a human IgG1 or IgG4 heavy chain constant region. In some embodiments, the anti-MUC16x anti-CD3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3. An anti-MUC16 x anti-CD3 bispecific antibody having a first antigen-binding domain that specifically binds to human MUC16 and comprises a HCVR comprising the amino acid sequence of SEQ ID NO:4, and a LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds to human CD3 and comprises a HCVR comprising the amino acid sequence of SEQ ID NO:5, and a LCVR comprising the amino acid sequence of SEQ ID NO:6, is referred to herein as mAb1.This antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO:1 (including an HCVR that specifically binds human MUC16), a second heavy chain comprising the amino acid sequence of SEQ ID NO:2 (including an HCVR that specifically binds human CD3), and a common light chain comprising the amino acid sequence of SEQ ID NO:3. In some cases, the mature forms of the antibodies may not include the C-terminal lysine residues of SEQ ID NOs:1 and 2. Thus, in some cases, the anti-MUC16 binding arm of mAb1 comprises a heavy chain comprising residues 1-442 of SEQ ID NO:1, and the anti-CD3 binding arm of mAb1 comprises a heavy chain comprising residues 1-449 of SEQ ID NO:2.

[0066] The amount of antibody or antigen-binding fragment thereof contained within the pharmaceutical formulation of the present invention can vary depending on the specific desired properties of the formulation and the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation can contain about 0.1 mg / mL to about 500 mg / mL of antibody, about 0.5 mg / mL to about 400 mg / mL of antibody, about 1 mg / mL to about 200 mg / mL of antibody, about 2 mg / mL to about 100 mg / mL, about 1 mg / mL to about 5 mg / mL of antibody, about 10 mg / mL to about 30 mg / mL of antibody, about 75 mg / mL to about 125 mg / mL, about 5 mg / mL to about 50 mg / mL, about 4 mg / mL to about 60 mg / ml, or about 2 mg / mL to about 55 mg / mL of antibody.For example, the formulations of the invention may comprise about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, approximately 18 mg / mL, approximately 19 mg / mL, approximately 20 mg / mL, approximately 21 mg / mL, approximately 22 mg / mL, approximately 23 mg / mL, approximately 24 mg / mL, approximately 25 mg / mL, approximately 26 mg / mL, approximately 27 mg / mL, approximately 28mg / mL, approximately 29mg / mL, approximately 30mg / mL, approximately 35mg / mL, approximately 40mg / mL, approximately 45mg / mL, approximately 50mg / mL, approximately 55mg / mL, approximately 60mg / mL, approximately 65mg / m L, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, Approximately 100mg / mL, Approximately 101mg / mL, Approximately 102mg / mL, Approximately 103mg / mL, Approximately 104mg / mL, Approximately 105mg / mL, Approximately 110mg / mL, Approximately 115mg / mL, Approximately 120mg / mL, Approximately 1 A liquid formulation comprising about 25 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, about 155 mg / mL, about 160 mg / mL, about 165 mg / mL, about 170 mg / mL, about 175 mg / mL, about 180 mg / mL, about 185 mg / mL, about 190 mg / mL, about 195 mg / mL, or about 200 mg / mL of an antibody, or antigen-binding fragment thereof. In certain embodiments, the pharmaceutical formulation is a liquid formulation that may contain 1±0.1 mg / mL to 200±20 mg / mL of antibody, 2±0.2 mg / mL to 10±1 mg / mL of antibody, 1±0.5 mg / mL to 30±5 mg / mL of antibody, 40±4 mg / mL to 60±6 mg / mL of antibody, 1±0.1 mg / mL to 3±0.3 mg / mL of antibody, 3±0.5 mg / mL to 7±0.5 mg / mL of antibody, 45±1 mg / mL to 55±1 mg / mL of antibody, 140±5 mg / mL to 160±5 mg / mL of antibody, or 175±5 mg / mL to 185±5 mg / mL of antibody.In some embodiments, the pharmaceutical formulation contains 5±0.5 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 50±5 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 150±15 mg / ml of antibody. In some embodiments, the pharmaceutical formulation contains 2±0.2 mg / ml of antibody. In some embodiments, the pharmaceutical formulation contains 20±2 mg / ml of antibody. In some embodiments, the pharmaceutical formulation contains 180±10 mg / ml of antibody.

[0067] biological equivalent The present invention encompasses antibodies having amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to human MUC16 and human CD3. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to the biological activity of the antibodies discussed herein.

[0068] The present invention includes antigen-binding molecules that are biologically equivalent to any of the exemplary antibodies described herein. Two antibodies are considered biologically equivalent if they are pharmaceutical equivalents or pharmaceutical substitutes, e.g., when administered in the same molar dose under similar experimental conditions, either in a single dose or multiple doses, and the rate and extent of absorption do not show significant differences. Some antibodies are considered to be equivalent or pharmaceutical substitutes if their degree of absorption, but not their rate of absorption, is equivalent, and furthermore, such differences in absorption rate are intentional, reflected in labeling, and are not essential for obtaining effective body drug concentrations in chronic use, and are not considered medically significant for the particular pharmaceutical product being studied, so they may be considered biologically equivalent.

[0069] In one embodiment, two antibodies are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0070] In one embodiment, two antibodies are bioequivalent if a patient can be switched between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to continued therapy without such switching.

[0071] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests in humans or other mammals in which the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo tests in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0072] Formulation excipients and pH The pharmaceutical formulations of the present invention include one or more excipients. As used herein, the term "excipient" refers to any non-therapeutic agent added to a formulation to provide a desired consistency, viscosity, or stabilizing effect.

[0073] In certain embodiments, the pharmaceutical formulation of the present invention includes one or more carbohydrates, such as one or more sugars. The sugar can be a reducing sugar or a non-reducing sugar. "Reducing sugar" includes, for example, sugars with a ketone or aldehyde group, and contains a reactive hemiacetal group that allows the sugar to act as a reducing agent. Specific examples of reducing sugars include fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose, and maltose. Non-reducing sugars are acetals and can contain an anomeric carbon that is not substantially reactive with amino acids or polypeptides to initiate the Maillard reaction. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, melezitose, and raffinose. Sugar acids include, for example, sugar acids, gluconates, and other polyhydroxy sugars, and their salts. In some embodiments, the sugar is sucrose. In some cases, the sugar (eg, sucrose) acts as a thermal stabilizer for the anti-MUC16 x anti-CD3 bispecific antibody.

[0074] The amount of sugar (e.g., sucrose) contained within the pharmaceutical formulation of the present invention will vary depending on the specific context and intended purpose for which the formulation is used. In certain embodiments, the formulation may contain about 0.1% to about 20% sugar, about 0.5% to about 20% sugar, about 1% to about 20% sugar, about 2% to about 15% sugar, about 5% to about 15% sugar, about 7.5% to about 12.5% ​​sugar, or about 9% to about 11% sugar. For example, the pharmaceutical formulations of the present invention may contain about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%, about 13.5%, about 14.0%, about 14.5%, about 15%, or about 20% sugar (e.g., sucrose). In some embodiments, the formulation contains about 10% sugar (e.g., sucrose). In some embodiments, the formulation contains about 5% sugar (e.g., sucrose). Each of the above percentages corresponds to a weight / volume (w / v) percentage. In some cases, the formulation contains 5%±1% to 20%±4% w / v sucrose. In some cases, the formulation contains 5% to 10% w / v sucrose. In some cases, the formulation contains 8%±0.5% to 12%±0.5% w / v sucrose. In some cases, the formulation contains 10%±1% w / v sucrose.

[0075] The pharmaceutical formulations of the invention may also include one or more organic co-solvents (or interfacial stabilizers) of a type and amount that stabilizes the anti-MUC16 x anti-CD3 bispecific antibody under conditions of rough handling or agitation, such as orbital shaking. In some embodiments, the organic co-solvent is a surfactant. As used herein, the term "surfactant" refers to a substance that reduces the surface tension of the fluid in which it is dissolved and / or reduces the interfacial tension between oil and water. Surfactants can be ionic or non-ionic. Exemplary non-ionic surfactants that may be included in the formulations of the invention include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific non-ionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85, poloxamers such as poloxamer 188 (also known as Pluronic F68), poloxamer 407, polyethylene polypropylene, or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate, and polyoxyethylene sorbitan monolaurate. In some embodiments, the surfactant is polysorbate 20.

[0076] The amount of surfactant contained within the pharmaceutical formulation of the present invention can vary depending on the specific desired properties of the formulation, as well as the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the formulation can contain from about 0.01% to about 1% surfactant, from about 0.01% to about 0.5% surfactant, from about 0.1% to about 0.3%, from about 0.15% to about 0.25% surfactant, or from about 0.19% to about 0.21% surfactant. For example, the formulations of the present invention may contain about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, or about 0.30% surfactant (e.g., polysorbate 20). In some embodiments, the formulation contains about 0.2% of a surfactant (e.g., polysorbate 20). In some embodiments, the formulation contains about 0.05% of a surfactant (e.g., polysorbate 20). Each of the above percentages corresponds to a weight / volume (w / v) percentage. In some cases, the formulation contains 0.01%±0.005% to 0.5%±0.25% w / v of polysorbate 20. In some cases, the formulation contains 0.2%±0.05% w / v of polysorbate 20. In some cases, the formulation contains 0.2%±0.01% w / v of polysorbate 20.

[0077] The pharmaceutical formulations of the invention may also include a buffer or buffering system that serves to maintain a stable pH and aid in stabilization of the anti-MUC16 x anti-CD3 bispecific antibody. In some embodiments, the buffer or buffering system includes at least one buffer having a buffer range that overlaps completely or partially with the pH 4.5 to 5.5 range. In certain embodiments, the buffer includes an acetate buffer (e.g., sodium acetate). In certain embodiments, the buffer (e.g., acetate) is present at a concentration of about 1 mM to about 50 mM, about 20 mM to about 40 mM, about 25 mM to about 35 mM, about 28 mM to about 32 mM, or about 29 mM to about 31 mM. In some embodiments, the buffer (e.g., acetate) is present at a concentration of about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, or about 40 mM. In some cases, the buffer is a histidine buffer present at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM. In some cases, the formulation contains a histidine buffer at a concentration of 5 mM ± 1 mM to 15 mM ± 3 mM. In some embodiments, the formulation contains an acetate buffer (e.g., at any of the concentrations described above or discussed herein). In some embodiments, the formulation contains a phosphate buffer (e.g., at any of the concentrations described above or discussed herein).

[0078] In some embodiments, the pharmaceutical formulation of the invention may also include arginine. Optionally, arginine is present at a concentration of 1-100 mM. In some embodiments, arginine is present at a concentration of 25-75 mM. Optionally, arginine is present at a concentration of 50 mM±5 mM. In one embodiment, the pharmaceutical formulation includes 30 mM±3 mM acetate, 7%±0.7% w / v sucrose, 0.05%±0.01% w / v polysorbate (e.g., polysorbate 20), and 50 mM±5 mM arginine at pH 5.0±0.1. Optionally, the antibody is present at a concentration of 1 mg / ml to 200 mg / ml, or 150 mg / ml±10 mg / ml.

[0079] During the antibody purification process, it may be desirable or necessary to exchange one buffer for another to achieve appropriate excipient concentrations, antibody concentrations, pH, etc. Buffer exchange can be accomplished, for example, by ultrafiltration / diafiltration (UF / DF), using, for example, semi-permeable tangential flow filtration membranes. However, the use of such techniques can give rise to the Gibbs-Donnan effect (Bolton et al., 2011, Biotechnol. Prog. 27(1):140-152). The accumulation of positive charges on the product side of the membrane during protein concentration is electrically counterbalanced by the preferential migration of positive ions to the other side of the membrane. A potential consequence of this phenomenon is that the final concentrations of certain components (e.g., acetate) may be lower than the intended target concentrations of these components due to electrostatic repulsion of positively charged diafiltration buffer excipients against the positively charged antibody protein during the UF / DF step. Thus, the present invention includes formulations in which the concentration of acetate, for example, differs from the amounts or ranges recited herein due to the Gibbs-Donnan effect.

[0080] Volume exclusion describes the behavior of a highly concentrated sample in which a large proportion of the total volume of the solution is taken up by solutes, particularly large molecules such as proteins, excluding solvent from this space. This then reduces the total volume of solvent available for other solutes to be dissolved, which can result in uneven distribution across the ultrafiltration membrane. Thus, the present invention includes formulations in which, for example, the concentration of acetate may differ from the amounts or ranges listed herein due to volume exclusion effects.

[0081] During the manufacture of the formulations of the invention, variations in the composition of the formulation may occur. These variations may include the concentration of the active ingredient, the concentration of the excipients, and / or the pH of the formulation. Thus, the invention includes formulations comprising anti-MUC16 x anti-CD3 bispecific antibodies that are stable and retain potency with up to at least 10% variation in excipient concentration. For example, included herein are anti-MUC16 x anti-CD3 bispecific antibodies, where the stability and potency of the formulation is not affected by ±10% or ±20% variations in the concentration of the antibody, sucrose, acetate buffer, and / or polysorbate.

[0082] Stability of pharmaceutical formulations The pharmaceutical formulation of the present invention exhibits a high level of stability. The term "stable" as used herein in relation to a pharmaceutical formulation means that the antibody in the pharmaceutical formulation retains an acceptable degree of structure and / or function and / or biological activity after storage for a specified amount of time. A formulation can be stable even if the antibody contained therein does not retain 100% of its structure and / or function and / or biological activity after storage for a specified amount of time. Under certain circumstances, retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure and / or function and / or biological activity of the antibody after storage for a specified amount of time can be considered "stable".

[0083] Stability can be measured, among other things, by determining the percentage of native antibody remaining in the formulation after storage at a given temperature for a specified amount of time. The percentage of native antibody can be determined, among other things, by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). "Acceptable degree of stability," as the phrase is used herein, means that at least 90% native form of the antibody can be detected in the formulation after storage at a given temperature for a specified amount of time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% native form of the antibody can be detected in the formulation after storage at a given temperature for a specified amount of time. The defined amount of time after which stability is measured can be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation may be stored when assessing stability can be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, the pharmaceutical formulation can be considered stable if greater than about 90%, 95%, 96%, or 97% of the native antibody is detected by SE-HPLC after 3 months of storage at 5°C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native antibodies are detected by SE-HPLC after 6 months of storage at 5° C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibodies are detected by SE-HPLC after 9 months of storage at 5° C.A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibodies are detected by SE-HPLC after 12 months of storage at 5° C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibodies are detected by SE-HPLC after 24 months of storage at 5° C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibodies are detected by SE-HPLC after 36 months of storage at 5° C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibody is detected by SE-UPLC after 3 months of storage at 25° C. (and optionally 60% relative humidity). A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibody is detected by SE-HPLC after 6 months of storage at 25° C. (and optionally 60% relative humidity). A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% of the native antibodies are detected by SE-HPLC after 9 months of storage at 25° C. (and optionally 60% relative humidity). A pharmaceutical formulation may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native antibodies are detected by SE-HPLC after 3 months of storage at 37° C. A pharmaceutical formulation may also be considered stable if greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the native antibodies are detected by SE-HPLC after 1 month of storage at 45° C. (and optionally 75% relative humidity).

[0084] Other methods, such as differential scanning calorimetry (DSC) to determine thermal stability, controlled stirring to determine mechanical stability, and absorbance at about 350 nm or about 405 nm to determine solution turbidity, can be used to assess the stability of the formulations of the invention. For example, the formulations of the invention may exhibit a OD of 0.18 to 0.25 g / ml after storage at about 5° C. to about 25° C. for 6 months or more. 405 The change in OD of the formulation at t=0 405 to less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less).

[0085] Measuring the binding affinity of an antibody to its target can also be used to assess stability. For example, a formulation of the invention can be considered stable if, after storage at, e.g., -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 45°C, etc., for a defined amount of time (e.g., 14 days to 9 months), the anti-MUC16 x anti-CD3 bispecific antibody contained within the formulation binds to human MUC16 and human CD3 with an affinity that is at least 80%, 85%, 90%, 95%, or more of the binding affinity of the antibody prior to such storage. Binding affinity can be determined by any method, e.g., ELISA or surface plasmon resonance. Biological activity can be determined by MUC16 or CD3 activity assays, such as by contacting cells expressing MUC16 or CD3 with a formulation comprising an anti-MUC16 x anti-CD3 bispecific antibody. Binding of the antibody to such cells can be measured directly, such as via FACS analysis.

[0086] Stability can be measured, among other things, by determining the percentage of antibodies that form aggregates (high molecular weight (HMW) species) in the formulation after storage at a specified temperature for a specified amount of time, with stability being inversely proportional to the percentage of aggregates formed. The percentage of aggregated antibodies can be determined, among other things, by size-exclusion chromatography (e.g., size-exclusion high performance liquid chromatography [SE-HPLC] or size-exclusion ultra performance liquid chromatography [SE-UPLC]). "Acceptable degree of stability," as the phrase is used herein, means that at most 6% of the antibodies are in aggregated form detectable in the formulation after storage at a given temperature (up to 25°C) for a specified amount of time. In certain embodiments, acceptable degree of stability means that at most about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibodies can be detected in aggregates in the formulation after storage at a given temperature for a specified amount of time. The specified amount of time after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature between about -80°C and about 45°C, for example, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. For example, a pharmaceutical formulation may be considered stable if less than about 3%, 2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibodies are detected in aggregated form after 12 months of storage at 5° C. In some cases, a pharmaceutical formulation may be considered stable if less than about 5%, 4.75%, 4.5%, 4.25%, 4%, 3.75%, 3.5%, 3.25%, 2%, or 1% of the antibodies are detected in aggregated form after 6 months of storage at 5° C.A pharmaceutical formulation may also be considered stable if less than about 6%, 5.75%, 5.5%, 5.25%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibodies are detected in aggregated form after 6 months of storage at 25° C. and 60% relative humidity. A pharmaceutical formulation may also be considered stable if less than about 6%, 5.75%, 5.5%, 5.25%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibodies are detected in aggregated form after 3 months of storage at 37° C. A pharmaceutical formulation may also be considered stable if less than about 3%, 2.75%, 2.5%, 2.25%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1%, 0.5%, or 0.1% of the antibody is detected in aggregated form after 12 months of storage at -30°C or -80°C.

[0087] Stability can be measured, inter alia, by determining the percentage of antibody remaining in the form of glycosylated species. The percentage of "glycosylated species" of an antibody can be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC] or cation exchange ultra performance liquid chromatography [CEX-UPLC]) and / or LC-MS. "Acceptable degree of stability," as the phrase is used herein, means that the percentage of antibody in the form of "glycosylated species" does not change by more than a specified amount after storage at a specified temperature for a specified amount of time. In certain embodiments, acceptable degree of stability means that the percentage of "glycosylated species" changes by 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, or 1% or less after storage at a given temperature for a specified amount of time. The specified amount of time after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if the rate of change in "glycated species" is 5% or less, 4% or less, 3% or less, 2% or less, or 1.5% or less after 12 months of storage at -80°C or -30°C. In another example, a pharmaceutical formulation may be considered stable if the rate of change of "glycated species" is 5% or less, 4% or less, 3% or less, 2% or less, or 1.5% or less after 6 months of storage at 5° C. In another example, a pharmaceutical formulation may be considered stable if the rate of change of "glycated species" is 5% or less, 4% or less, or 3% or less after 12 months of storage at 5° C.In each case, the measurements may be carried out using cation exchange ultra performance liquid chromatography (CEX-UPLC) and / or by LC-MS.

[0088] Reference to the stability of a pharmaceutical formulation "after" a specified period of time is intended to mean that a measurement of the stability parameter (e.g., % native, % HMW species, or % acidic) is made at or near the end of the specified period of time, and is not intended to mean that the pharmaceutical formulation will necessarily maintain the same degree of stability for the parameter measured thereafter. For example, a reference to a particular stability after 12 months means that the stability measurement is made at or near 12 months after the start of the study. Additional methods for assessing the stability of an antibody in a formulation are demonstrated in the Examples provided below.

[0089] As illustrated in the Examples below, the present invention is based, in part, on the discovery that the combination of the claimed excipients with a bispecific anti-MUC16 x anti-CD3 antibody produces formulations that are stable.

[0090] Exemplary Formulations According to one aspect of the invention, a pharmaceutical formulation comprises (i) a human anti-MUC16×anti-CD3 bispecific antibody that specifically binds to human MUC16 and human CD3, (ii) a buffer comprising an acetate salt (e.g., sodium acetate), (iii) an organic co-solvent comprising a polysorbate, and (iv) a stabilizer comprising a sugar. According to another aspect, a pharmaceutical formulation comprises (i) a human anti-MUC16×anti-CD3 bispecific antibody that specifically binds to human MUC16 and human CD3, (ii) a buffer comprising an acetate salt, and (iii) a stabilizer comprising a sugar. According to another aspect of the invention, a pharmaceutical formulation comprises (i) a human anti-MUC16×anti-CD3 bispecific antibody that specifically binds to human MUC16 and human CD3, (ii) a buffer comprising histidine, (iii) an organic co-solvent comprising a polysorbate, and (iv) a stabilizer comprising a sugar.

[0091] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 4 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6, at a concentration of about 1 mg / ml to about 200 mg / ml, and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 5 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6; (ii) acetate at a concentration of about 25 mM to about 35 mM; (iii) polysorbate 20 at a concentration of about 0.1% w / v to about 0.3% w / v; and (iv) sucrose at a concentration of about 5% w / v to about 15% w / v, wherein the formulation has a pH of 5.0±0.3.

[0092] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of about 1 mg / ml to about 200 mg / ml, comprising: a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 25 mM to about 35 mM; (iii) polysorbate 20 at a concentration of about 0.1% w / v to about 0.3% w / v; and (iv) sucrose at a concentration of about 5% w / v to about 15% w / v, wherein the formulation has a pH of 5.0±0.3.

[0093] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of about 1 mg / ml to about 200 mg / ml, comprising: a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 25 mM to about 35 mM; (iii) polysorbate 20 at a concentration of about 0.1% w / v to about 0.3% w / v; and (iv) sucrose at a concentration of about 5% w / v to about 15% w / v, wherein the formulation has a pH of 5.0±0.3.

[0094] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3, at a concentration of about 1 mg / ml to about 200 mg / ml; (ii) acetate at a concentration of about 25 mM to about 35 mM; (iii) polysorbate 20 at a concentration of about 0.1% w / v to about 0.3% w / v; and (iv) sucrose at a concentration of about 5% w / v to about 15% w / v, wherein the formulation has a pH of 5.0±0.3.

[0095] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4, and a LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5, and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0096] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of about 1 mg / ml to about 200 mg / ml, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0097] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of about 1 mg / ml to about 200 mg / ml, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0098] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3, at a concentration of about 1 mg / ml to about 200 mg / ml; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0099] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, in a concentration of about 1 mg / ml to about 200 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v; and (iv) sucrose at a concentration of about 10% w / v, wherein the formulation has a pH of 5.0±0.1.

[0100] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific IgG1A antibody, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of about 1 mg / ml to about 200 mg / ml. (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v; and (iv) sucrose at a concentration of about 10% w / v, wherein the formulation has a pH of 5.0±0.1.

[0101] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific IgG1A antibody, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of about 1 mg / ml to about 200 mg / ml. (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v; and (iv) sucrose at a concentration of about 10% w / v, wherein the formulation has a pH of 5.0±0.1.

[0102] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3, at a concentration of about 1 mg / ml to about 200 mg / ml; (ii) histidine at a concentration of 30 mM; (iii) polysorbate 20 at a concentration of 0.2% w / v; and (iv) sucrose at a concentration of about 10% w / v, wherein the formulation has a pH of 5.0±0.1.

[0103] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, in a concentration of 3 mg / ml ± 1 mg / ml to 7 mg / ml ± 1 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0104] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4, and a LCVR comprising the amino acid sequence of SEQ ID NO:6, in a concentration of 5 mg / ml ± 0.5 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5, and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0105] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, in a concentration of 40 mg / ml ± 2 mg / ml to 60 mg / ml ± 2 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0106] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, in a concentration of 50 mg / ml ± 5 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0107] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of 5 mg / ml ± 0.5 mg / ml, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0108] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody, at a concentration of 50 mg / ml ± 5 mg / ml, comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0109] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of 5 mg / ml ± 0.5 mg / ml, wherein the antibody has a heavy chain constant region of isotype IgG4, one of the two heavy chains being unmodified IgG4 (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0110] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen-binding domain that specifically binds human CD3, the second antigen-binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of 50 mg / ml ± 0.5 mg / ml, wherein the antibody has a heavy chain constant region of isotype IgG4, one of the two heavy chains being unmodified IgG4 (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0111] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3, at a concentration of 5 mg / ml ± 0.5 mg / ml; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0112] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO:1, a second heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a common light chain comprising the amino acid sequence of SEQ ID NO:3, at a concentration of 50 mg / ml ± 5 mg / ml; (ii) acetate at a concentration of about 30 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.2% w / v ± 0.02% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0113] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of about 1 mg / ml to about 3 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) histidine at a concentration of about 10 mM ± 1 mM; (iii) polysorbate 20 at a concentration of about 0.05% w / v ± 0.01% w / v; and (iv) sucrose at a concentration of about 10% w / v ± 1% w / v, wherein the formulation has a pH of 6.0 ± 0.3.

[0114] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, at a concentration of about 150 mg / ml to about 200 mg / ml; and a second antigen-binding domain that specifically binds human CD3, the first antigen-binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO:6; (ii) acetic acid at a concentration of about 30 mM ± 1 mM; and (iii) sucrose at a concentration of about 5% w / v ± 0.5% w / v, wherein the formulation has a pH of 5.0 ± 0.3.

[0115] For any of these exemplary formulations, "stable" may be defined as follows: (a) the formulation contains 2.5% or less high molecular weight (HMW) species after 12 months of storage at 5° C., as determined by SE-UPLC; (b) the formulation contains 3.5% or less high molecular weight (HMW) species after 6 months of storage at 25° C. and 60% relative humidity, as determined by SE-UPLC; (c) the formulation contains 3.5% or less high molecular weight (HMW) species after 12 months of storage at −30° C., as determined by SE-UPLC. (d) the formulation contains 1.5% or less high molecular weight (HMW) species after 12 months storage at -80°C as determined by SE-UPLC; (e) at least 95% of the antibody has native conformation after 12 months storage at 5°C as determined by SE-UPLC; (f) at least 95% of the antibody has native conformation after 6 months storage at 25°C and 60% relative humidity as determined by SE-UPLC. (g) at least 95% of the antibody is in the native conformation after 3 months storage at 37° C. as determined by SE-UPLC; (h) the formulation contains 1% or less high molecular weight (HMW) species after 12 months storage at 5° C. as determined by SE-UPLC; (i) the formulation contains 1% or less HMW species after 6 months storage at 25° C. and 60% relative humidity as determined by SE-UPLC; (j) the formulation contains 1% or less HMW species after 6 months storage at 37° C. and 60% relative humidity as determined by SE-UPLC. (k) the formulation contains 2% or less high molecular weight (HMW) species after 12 months storage at -30°C or -80°C as determined by SE-UPLC; (l) the formulation contains 4% or less HMW species after 6 months storage at 5°C as determined by SE-UPLC; or (m) the formulation contains 6% or less HMW species after 6 months storage at 25°C and 60% relative humidity as determined by SE-UPLC.

[0116] In any of these exemplary formulations, the bispecific antibody may comprise a modification in one or both heavy chains to facilitate purification of the bispecific antibody (i.e., heterodimer) from homodimeric impurities. In some embodiments, the bispecific antibody comprises first and second heavy chains (i.e., a heavy chain of an anti-MUC16 binding arm and a heavy chain of an anti-CD3 binding arm) that are identical (e.g., both are isotype IgG1 or IgG4) except for a modification in the CH3 domain of one or the other heavy chain that reduces binding of the bispecific antibody to Protein A compared to an antibody lacking the modification. In some cases, the CH3 domain of the first heavy chain (e.g., of the anti-MUC16 binding arm) binds Protein A and the CH3 domain of the second heavy chain (e.g., of the anti-CD3 binding arm) comprises a mutation that reduces or abolishes Protein A binding. In some cases, the mutation is an H435R modification (according to EU numbering; H95R according to IMGT exon numbering). In some cases, the mutations are H435R modifications (according to EU numbering; IMGT exon numbering H95R) and Y436F modifications (according to EU numbering; Y96F by IMGT). Further modifications that may be found in the second CH3 domain include: D356E, L358M, N384S, K392N, V397M, and V422I by EU for IgG1 CH3 domains (D16E, L18M, N44S, K52N, V57M, and V82I by IMGT), and Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU (Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I by IMGT) for IgG4 CH3 domains.

[0117] Additional non-limiting examples of pharmaceutical formulations encompassed by the present invention are described elsewhere herein, including in the Examples provided below.

[0118] Container and method of administration The pharmaceutical formulations of the present invention can be contained in any container suitable for storing pharmaceuticals and other therapeutic compositions. For example, the pharmaceutical formulations can be contained in a sealed and sterilized plastic or glass container having a predetermined volume, such as a vial, an ampoule, a syringe, a cartridge, a bottle, or an IV bag. Different types of vials, including, for example, clear and opaque (e.g., tan) glass or plastic vials, can be used to contain the formulations of the present invention. Similarly, any type of syringe can be used to contain and / or administer the pharmaceutical formulations of the present invention. In some embodiments, the pharmaceutical formulations are contained in a pre-filled syringe. In some embodiments, the pharmaceutical formulations are contained in a pre-filled staked needle syringe.

[0119] The pharmaceutical formulations of the present invention may be contained within "normal tungsten" or "low tungsten" syringes. As will be appreciated by those skilled in the art, the process of making glass syringes generally involves the use of a hot tungsten rod that functions to pierce the glass, thereby creating a hole through which liquid can be drawn and expelled from the syringe. This process deposits trace amounts of tungsten on the interior surface of the syringe. Subsequent cleaning and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means that the syringe contains greater than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe according to the present invention can contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 ppb or less of tungsten.

[0120] Rubber plungers used in syringes and rubber stoppers used to close the openings of vials can be coated to prevent contamination of the pharmaceutical contents of the syringe or vial and / or to preserve their stability. Thus, the pharmaceutical formulations of the present invention according to certain embodiments can be contained in a syringe with a coated plunger or in a vial sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of coated stoppers and / or plungers suitable for use with vials and syringes containing the pharmaceutical formulations of the present invention are mentioned, for example, in U.S. Patent Nos. 4,997,423, 5,908,686, 6,286,699, 6,645,635, and 7,226,554, the contents of which are incorporated herein by reference in their entirety. Certain exemplary coated rubber stoppers and plungers that may be used in the context of the present invention are commercially available under the trademark "FluroTec®" available from West Pharmaceutical Services, Inc. (Lionville, PA). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent pharmaceuticals from adhering to rubber surfaces. According to certain embodiments of the present invention, the pharmaceutical formulation may be contained within a low tungsten syringe with a fluorocarbon coated plunger. In some embodiments, the container is a syringe, such as an Ompi EZ-Fill™ syringe or a BD Neopak™ syringe. In some cases, the syringe is a 1 mL long glass syringe with a 1 mL iWest piston, a 27G thin wall needle, and an FM30 needle shield or a BD260 needle shield. In some cases, the syringe is a 2.25 mL glass syringe with a West NovaPure™ 1-3 mL piston, a 27G thin wall needle, and an FM30 needle shield or a BD260 needle shield.In various embodiments, the syringe is a 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, 8.0 mL, 8.5 mL, 9.0 mL, 9.5 mL, or 10 mL syringe (e.g., a glass syringe).

[0121] The pharmaceutical formulations can be administered to a patient by parenteral routes, such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or by transdermal, mucosal, nasal, pulmonary, and / or oral administration. A number of reusable pen and / or autoinjector delivery devices can be used to deliver the pharmaceutical formulations of the invention subcutaneously. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN ... STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen and / or autoinjector delivery devices that have application in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name just a few. In some cases, the pharmaceutical formulation is contained in a syringe that is specifically adapted for use with the autoinjector.Subcutaneous injections may be administered using a 20-30 gauge needle, or a 25-30 gauge needle. In some cases, subcutaneous injections may be administered using a 25 gauge needle. In some cases, subcutaneous injections may be administered using a 27 gauge needle. In some cases, subcutaneous injections may be administered using a 29 gauge needle.

[0122] Another type of delivery device can include a safety system. Such devices can be relatively inexpensive and can be operated to manually or automatically extend a safety sleeve over the needle once the injection is complete. Examples of safety systems can include the ERIS device by West Pharmaceutical, or the UltraSafe device by Becton Dickinson. Additionally, the use of a large volume device ("LVD") or bolus injector to deliver the pharmaceutical formulation of the present invention is also contemplated herein. In some cases, the LVD or bolus injector can be configured to inject the drug into the patient. For example, the LVD or bolus injector can be configured to deliver a "large" volume of the drug (typically about 2 ml to about 10 ml).

[0123] In certain embodiments, the pharmaceutical formulation is administered via intravenous infusion, such that the formulation is diluted in an IV bag containing a physiologically acceptable solution. In one embodiment, the pharmaceutical composition is a composite sterile preparation in an intravenous infusion bag, such that a single dose of pharmaceutical product is diluted in 100 mL, 250 mL (or other similar volume suitable for intravenous infusion delivery) of a physiological buffer solution (e.g., 0.9% saline).

[0124] The pharmaceutical formulations of the present invention may also be contained in unit dosage forms. The term "unit dosage form" as used herein refers to physically discrete units suitable as single dosages for the patient to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, diluent, or excipient. In various embodiments, the unit dosage form is contained within a container as discussed herein. The actual dosage level of the active ingredient (e.g., anti-MUC16 x anti-CD3 bispecific antibody) in the formulation of the present invention may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for the particular patient, composition, and mode of administration without adversely affecting the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts. The term "diluent" as used herein refers to a solution suitable for modifying or achieving one or more of the exemplary or suitable concentrations described herein.

[0125] In various embodiments, the unit dosage form contains the amount of active ingredient (e.g., anti-MUC16 x anti-CD3 bispecific antibody) intended for a single use. In various embodiments, the amount of active ingredient in the unit dosage form is about 0.1 mg to about 5000 mg, about 100 mg to about 1000 mg, and about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 200 mg, about 40 mg to about 60 mg, about 125 mg to about 175 mg, about 160 mg to about 200 mg, about 1 mg to about 250 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg, about 1 mg to about 20 mg, about 5 mg to about 15 mg, or ranges or intervals therein. Intermediate ranges between the amounts recited above, such as about 2 mg to about 100 mg or 2 mg to 20 mg, are also contemplated as part of the invention. For example, ranges of values ​​using any combination of the values ​​recited above (or values ​​included within the ranges recited above) as upper and / or lower limits are intended to be included. In some embodiments, the unit dosage form contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg of antibody. In some embodiments, the unit dosage form contains 5 mg of antibody. In some embodiments, the unit dosage form contains 12.5 mg of antibody. In certain embodiments, the formulation is often supplied as a liquid in unit dosage form. In some embodiments, the unit dosage form contains 3-7 mg, or 8-12 mg, 10-15 mg, 35-45 mg, 45-55 mg, 140-160 mg, or 170-190 mg. In some embodiments, the unit dosage form according to the invention is suitable for subcutaneous administration to a patient (e.g., a unit dosage form containing an antibody at a concentration of about 100 mg / ml or about 200 mg / ml, or 150 mg / ml ± 5 mg / ml).

[0126] The present invention also includes a method of preparing a unit dosage form. In an exemplary embodiment, the method for preparing a pharmaceutical unit dosage form includes combining the formulations of any of the above embodiments in a suitable container (e.g., a container discussed herein).

[0127] Therapeutic Uses of Pharmaceutical Preparations The pharmaceutical formulations of the present invention are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder associated with cells expressing human MUC16. Exemplary non-limiting diseases and disorders that can be treated by administration of the pharmaceutical formulations of the present invention include ovarian cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer.

[0128] The therapeutic methods of the invention include administering to a subject any of the formulations comprising the anti-MUC16 x anti-CD3 bispecific antibodies disclosed herein. The subject to whom the pharmaceutical formulation is administered may, for example, be any human or non-human animal in need of such treatment. For example, the subject may be an individual diagnosed with or considered at risk for any of the aforementioned diseases or disorders. The invention further includes the use of any of the pharmaceutical formulations disclosed herein in the manufacture of a medicament for the treatment of any disease or disorder associated with cells expressing human MUC16, including any of the exemplary diseases, disorders, and conditions described above.

[0129] In some embodiments, the invention provides kits that include a pharmaceutical formulation (e.g., a container containing a formulation or unit dosage form) discussed herein and packaging or labeling (e.g., a package insert) that includes instructions for using the pharmaceutical formulation for the treatment of a disease or disorder discussed above. In some cases, the instructions provide for the use of the unit dosage form discussed herein for the treatment of a disease or disorder.

[0130] A summary of the sequences referred to herein and their corresponding SEQ ID NOs is provided in Table 1 below. [Table 1] EXAMPLES

[0131] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0132] Example 1: Development of stable liquid and lyophilized anti-MUC16 x anti-CD3 bispecific antibody formulations Physical stability of a formulation refers to properties such as color, appearance, pH, turbidity, particles, and protein concentration. Chemical stability refers to the formation of high molecular weight (HMW) species, low molecular weight (LMW) species, charge variants, and other chemical modifications of the protein. The physical and chemical stability of the mAb1 drug products was evaluated using the following assays: color and appearance by visual inspection; pH; turbidity as measured by the increase in optical density (OD) at 405 nm; analysis of particles subvisible to the naked eye by Micro-Flow Imaging™ (MFI); protein concentration by reversed-phase ultra-performance liquid chromatography (RP-UPLC); purity of each individual drug product was assessed using size-exclusion ultra-performance liquid chromatography (SE-UPLC) and reducing and non-reducing microchip capillary electrophoresis-sodium dodecyl sulfate (MCESDS); charge variant analysis was determined using cation exchange UPLC (CEX-UPLC) (glycated mAb1 was detected by CEX-UPLC) and imaging capillary isoelectric focusing (iCIEF); and potency by bioassay (the relative potency of each sample was determined by bioassay and (IC 50 Reference sample / IC 50 Storage stability is defined as the percent potency of the reference standard × 100%, and the measured potency of the storage stability sample should be within 50-150% of the measured potency of the reference standard).

[0133] A lyophilized formulation of mAb1 was developed for intravenous (IV) or subcutaneous (SC) administration. The lyophilized mAb1 drug product can be reconstituted with sterile water for injection (WFI) to a concentration of 2 mg / mL mAb1 or 20 mg / ml mAb1 for IV infusion or SC injection. Formulation development activities involved evaluation of buffers, pH, organic co-solvents, surfactants, and sucrose (as a heat stabilizer) to identify excipients that would improve protein stability. Results generated from these studies were used to develop a stable lyophilized formulation that is suitable for reconstitution into a liquid form.

[0134] Buffer and pH Selection The effect of buffer and pH on the thermal stability of mAb1 was first examined in liquid formulations by incubating 2 mg / mL of mAb1 in a series of buffer systems at various pHs at 45 °C for 28 days. The following pH and buffer systems were studied: acetate (pH 4.5-5.5), histidine (pH 5.5-6.5), and phosphate (pH 6.5-7.5). These analyses revealed that the formation of HMW species and charge variants were the major degradation pathways. Based on the results from SE-UPLC, the lowest rates of HMW species formation and the lowest rates of monomer loss were observed when mAb1 was formulated in histidine buffer at pH 5.5-6.5 or in acetate buffer at pH 4.5-5.5, as shown in Table 2. CEX-UPLC analysis also showed that the charge variant profile was most stable when mAb1 was formulated in histidine buffer at pH 5.5-6.5 or in acetate buffer at pH 4.5-5.5, as shown in Table 2. Compared to the starting material, a decrease in the relative amount of total acidic species and a concomitant increase in the major charge variant peak was observed by CEX-UPLC analysis after incubation of mAb1 at 45° C. for 28 days.

[0135] A single peak was isolated that contained approximately 50% of the total acidic charge variants observed in the CEX-UPLC chromatogram. Further analysis identified the major component in this peak as mAb1 protein containing glycosylation of heavy chain (HC) Lys98 within the CDR3 of the MUC16 binding arm of the bispecific antibody (glycosylation is discussed in detail in the following paragraph). During early activities related to the development of the mAb1 formulation, the stability of the major charge variants, total acidic species, total basic species, and glycosylation forms of the molecule were all monitored. A histidine buffer at pH 6.0 was selected for the clinically viable formulation because the rates of HMW species formation, monomer loss, and charge variant formation were minimal in this buffer and pH.

[0136] A specific mAb1-derived acidic peak containing approximately 50% of the total mAb1-derived acidic charge variants was observed by CEX-UPLC analysis. This peak area detected by CEX-UPLC decreased after 28 days of incubation at 45°C. This peak was identified as a mAb1 variant containing glycosylation at HC Lys98 within the CDR3 of the MUC16-binding arm of the bispecific antibody. An increase in potency by bioassay was observed with a decrease in the level of glycoforms of mAb1 (see Figure 1). Although Lys98 deglycosylation occurs readily in solution, the level of glycosylation in the lyophilized state remained unchanged when the formulation was stored at 5°C for at least 12 months. In addition, potency remained unchanged when the lyophilized formulation was stored at 5°C for up to 12 months. Thus, the lyophilized formulation was selected as the formulation of mAb1.

[0137] Surfactant / organic cosolvent selection The effect of the surfactants polysorbate 20 and polysorbate 80 on the agitation stress and thermal stability of mAb1 at 5 mg / mL was examined in liquid formulations (10% sucrose was present in the surfactant-containing formulations). Polysorbate 20 and polysorbate 80 at concentrations of 0-0.1% (w / v) were tested for their ability to stabilize mAb1 (see Tables 3 and 4 below). Both polysorbate 20 and polysorbate 80 were able to stabilize mAb1 against agitation stress when present at levels of 0.01% or greater, as shown in Table 3. When no surfactant was included in the formulation, an increase in HMW species of up to 1.9% was observed. When ≧0.01% polysorbate 20 or polysorbate 80 was included in the formulation, no increase in HMW species was observed.

[0138] Concentrations of 0.05% polysorbate 20 and 0.05% polysorbate 80 were selected to examine the effect of heat stress on mAb1 stability. After 28 days of incubation at 45° C., approximately 0.5% less HMW species and 1.6% more mAb1 monomer were observed in the formulation containing 0.05% polysorbate 20 compared to the formulation containing 0.05% polysorbate 80, as shown in Table 4. Furthermore, mAb1 showed an improved stability profile in the presence of 0.05% polysorbate 20 as determined by CEX-UPLC analysis after 28 days of incubation at 45° C. Compared to mAb1 formulated in the presence of 0.05% polysorbate 80 (see Table 4), mAb1 in the presence of 0.05% polysorbate 20 showed (1) approximately a 5% reduction in the formation of acidic species, (2) a 6% reduction in the loss of the major charge variant form, and (3) a nearly comparable change in the level of glycosylated species. 0.05% polysorbate 20 was selected as the surfactant for mAb1 pharmaceutical formulation because it adequately stabilized the protein against agitation stress.

[0139] Heat stabilizer / freeze protectant Stabilizers such as sucrose can sometimes be added to liquid and lyophilized antibody formulations to increase the thermal stability and stability of proteins against freeze / thaw stress. Inclusion of sucrose in the mAb1 formulation was required to stabilize mAb1 against freeze / thaw stress. In the absence of sucrose, the level of HMW species increased by 0.4% after mAb1 underwent four freeze and thaw cycles (see Table 5). Addition of 10% sucrose to the formulation stabilized the mAb1 drug substance against four freeze / thaw cycles (see Table 5).

[0140] Sucrose was also required as a cryoprotectant to stabilize mAb1 during the lyophilization process. After reconstitution of the lyophilized mAb1 formulation, the relative amount of HMW species increased by 0.2% in the absence of sucrose, as shown in Table 6, but showed no apparent change when the formulation contained 10% sucrose. When lyophilized mAb1 was incubated at 50° C. for 28 days in the absence of sucrose, the level of HMW species increased by 5.9% and the level of monomer decreased by 7.1% as determined by SE-UPLC (see Table 7). Furthermore, after incubation of mAb1 at 50° C. in the absence of sucrose, the level of acidic species decreased by 13.8%, the level of basic species increased by 14.3%, and the level of glycated species decreased by 14.7% as determined by CEX-UPLC (see Table 7). However, in the presence of 10% sucrose, no obvious changes in the levels of molecular weight variants or charge variants were observed, as shown in Table 7.

[0141] 10 percent sucrose had a positive effect on the freeze / thaw stability, thermal stability, and stability of mAb1 during the lyophilization process. 10 percent sucrose is also used as a bulking agent during lyophilization. Therefore, 10% sucrose was selected as a stabilizer for the development of lyophilized mAb1 formulations.

[0142] Overview of selected formulation ingredients mAb1 showed maximum stability when formulated in the presence of 10 mM histidine, 0.050% polysorbate 20, and 10% sucrose at pH 6.0. The major degradation pathways identified during the development of the mAb1 liquid formulation were the formation of HMW species and charge variants. A particularly noteworthy observed charge variant is the glycosylation of HC-CDR3-Lys98. It has been demonstrated that the level of glycosylated mAb1 is unchanged after incubation of lyophilized mAb1 under stress conditions and real-time storage conditions. The information obtained during the liquid formulation development and lyophilization feasibility study formed the basis for developing a formulation for a lyophilized drug product suitable for clinical use. To allow for reconstitution of the lyophilized drug product to a concentration of 2 mg / mL mAb1 for IV administration, a formulated drug substance was developed containing 2 mg / mL mAb1, 10 mM histidine, 10% (w / v) sucrose, and 0.05% (w / v) polysorbate 20, pH 6.0. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4] [Table 5] [Table 6] [Table 7]

[0143] Example 2: Storage and stress stability of formulations A study was conducted to evaluate the storage, accelerated stability, and stress stability (agitation) of liquid, lyophilized, and reconstituted formulations of mAb1 drug product. Results from the analysis of the mAb1 lyophilized drug product indicate that the mAb1 drug product was physically and chemically stable when stored at 5° C. for at least 36 months (see Tables 8, 10, and 12). No obvious changes in physical or chemical stability were detected in any of the monitored attributes. Results from the analysis of the mAb1 lyophilized formulation after incubation under accelerated conditions are provided in Tables 9, 11, and 13. No obvious changes in physical or chemical stability of mAb1 were detected in any of the monitored attributes after 3 months of incubation at 37° C. Similarly, no obvious changes in physical or chemical stability were detected in any of the monitored attributes after 6 months of incubation at 25° C. / 60% relative humidity (RH). A minor increase in HMW species (0.3%) and basic species (7.6% by CEX-UPLC and 4.0% by icIEF) was observed after 3 months of incubation at 50° C. mAb1 maintained potency as determined by bioassay analysis after incubation under accelerated conditions. [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2] [Table 10-1] [Table 10-2] [Table 11-1] [Table 11-2] [Table 12-1] [Table 12-2] [Table 13-1] [Table 13-2]

[0144] Additional stability studies of the reconstituted mAb1 drug product were also conducted to evaluate the stability of the reconstituted drug product when incubated at 25° C. for up to 24 hours as well as under stress (agitation) conditions. Lyophilized drug product (2.5 mL of formulated drug substance in 5 mL type 1 glass vial) was reconstituted with 2.3 mL of WFI (to a final volume of 2.5 mL) to 2 mg / mL mAb1 or 20 mg / ml mAb1. The results of these stability studies are shown in Tables 14 and 15 below. Reconstituted mAb1 drug product solutions at 2 mg / mL mAb1 and 20 mg / ml mAb1 were found to be physically and chemically stable when incubated at 25° C. for 24 hours. No obvious changes in physical or chemical stability were detected in any of the attributes monitored. These data indicate that the reconstituted drug product is stable at room temperature. Reconstituted mAb1 drug product solutions at 2 mg / mL mAb1 and 20 mg / ml mAb1 were also found to be physically and chemically stable when agitated (vortexed at ambient temperature) for 60 minutes. No obvious changes in physical or chemical stability were detected in any of the attributes monitored. [Table 14-1] [Table 14-2] [Table 15-1] [Table 15-2]

[0145] Results from mAb1 drug product storage and stress stability studies indicate that mAb1 is stable. mAb1 formulations can withstand short-term exposure to room temperature without compromising physical or chemical stability. mAb1 formulations are also stable when reconstituted to concentrations of 2 mg / mL or 20 mg / ml. Exposure of the reconstituted mAb1 drug product to 25°C for up to 24 hours does not compromise protein integrity nor does agitation of the reconstituted drug product.

[0146] The reconstitution amounts of the mAb1 drug products are shown in Tables 16 and 17 below. [Table 16] [Table 17]

[0147] As discussed more fully below, exploratory stability studies were conducted to determine the long-term storage, accelerated stability (temperatures above storage conditions), and stress stability (40° C. / 75% RH, agitation, freeze and thaw) of mAb1 drug substance (≧180 mg / ml mAb1, 30 mM acetate, 5% w / v sucrose, pH 5.0) and formulated drug substance (50 mg / ml mAb1, 30 mM acetate, 10% w / v sucrose, 0.2% w / v polysorbate 20, pH 5.0).

[0148] No obvious changes in the physical or chemical stability of mAb1 drug substance were detected when stored at -80°C and -30°C for up to 24 months (see Tables 18 and 19). These results indicate that mAb1 drug substance is stable for at least 24 months when stored frozen at storage conditions. Results from the exploratory accelerated stability study are presented in Tables 20-22. After incubation at -20°C for up to 6 months, no obvious changes were observed in any of the monitored attributes. An increase in protein concentration was observed by SoloVPE after up to 6 months of incubation at 5°C and 25°C / 60%RH, likely due to sample evaporation. An increase in HMW species was observed by SE-UPLC and non-reduced MCE after up to 6 months of incubation at 5°C and 25°C / 60%RH. A decrease in region 1 (acidic species) with a concomitant increase in region 2 (major peak) was observed by CEX-UPLC after 6 months of incubation at 5° C. and 25° C. / 60% RH, due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. An increase in region 1 (acidic species) with a concomitant decrease in region 2 (major peak) was observed by iCIEF after 6 months of incubation at 25° C. / 60% RH, likely due to deamidation. These results indicate that the mAb1 drug substance can withstand at least 6 months of incubation at −20° C. without compromising either the physical or chemical stability of the protein. Results from the exploratory stress stability study are presented in Tables 23, 24, and 17. The mAb1 drug substance was physically and chemically stable when agitated (vortexed) for 10 minutes or exposed to four freeze / thaw cycles. An increase in protein concentration was observed by SoloVPE after up to 3 months of incubation at 40°C / 75%RH, likely due to sample evaporation. An increase in color intensity was observed after 3 months of incubation at 40°C / 75%RH. An increase in HMW and LMW species was observed by SE-UPLC and MCE after up to 3 months of incubation at 40°C / 75%RH.A decrease in region 1 with a concomitant increase in region 2 was observed by CEX-UPLC after incubation at 40°C / 75%RH due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. An increase in region 3 with a concomitant decrease in region 2 was observed by CEX-UPLC after 3 months of incubation at 40°C / 75%RH. The increase in region 3 is due to an increase in the basic peak eluted during the high salt elution step required for CEX-UPLC. Results from accelerated and stressed conditions indicated that HMW, LMW, and charge variants were the major degradation pathways for mAb1 drug substance. [Table 18-1] [Table 18-2] [Table 19-1] [Table 19-2] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24]

[0149] No obvious changes in the physical or chemical stability of mAb1 formulated drug substance were detected when stored at -80°C and -30°C for up to 24 months (see Tables 25 and 26). These results indicate that mAb1 formulated drug substance is stable for at least 24 months when stored frozen at storage conditions. Results from the exploratory accelerated stability study are presented in Tables 27-29. No obvious changes in monitored attributes were observed after incubation of mAb1 formulated drug substance at -20°C or 5°C for up to 6 months. An increase in protein concentration was observed by SoloVPE after up to 6 months of incubation at 25°C / 60% RH, likely due to sample evaporation. An increase in HMW species was observed by SE-UPLC and non-reduced MCE after up to 6 months of incubation at 5°C and 25°C / 60% RH. A decrease in region 1 (acidic species) with a concomitant increase in region 2 (major peak) was observed by CEX-UPLC after 6 months of incubation at 25°C / 60%RH due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. An increase in region 1 (acidic species) with a concomitant decrease in region 2 (major peak) was observed by iCIEF after 6 months of incubation at 25°C / 60%RH, likely due to deamidation. These results indicate that mAb1 formulated drug substance can withstand incubation at -20°C for at least 6 months and at 5°C for 3 months without compromising either the physical or chemical stability of the protein. mAb1 formulated drug substance can also withstand short-term exposure to temperatures of 25°C / 60%RH. Results from the exploratory stress stability study are presented in Tables 30 and 31. The mAb1 formulated drug substance was physically and chemically stable when agitated (vortexed) for up to 120 minutes or subjected to up to four cycles of freezing and thawing (a slight increase in particles was observed by MFI after undergoing four freeze / thaw cycles). An increase in protein concentration was observed by SoloVPE after up to three months of incubation at 40°C / 75%RH, likely due to sample evaporation. An increase in HMW and LMW species was observed by SE-UPLC and MCE after up to three months of incubation at 40°C / 75%RH.A decrease in region 1 with a concomitant increase in region 2 was observed by CEX-UPLC after incubation at 40°C / 75%RH due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. After 2 months of incubation at 40°C / 75%RH, there is a decrease in region 1 and a concomitant increase in region 2. However, after the 3 month time point, the trend is reversed and there is a clear increase in region 1 and region 3 with a concomitant decrease in region 2. The increase in region 1 is likely due to competitive deamidation on asparagine or glutamine, while the increase in region 3 is due to an increase in the basic peak eluted in the high salt elution step. Results from accelerated and stressed conditions indicated that HMW, LMW, and charge variants were the major degradation pathways for mAb1 formulated drug substance. A slight increase in 2-10 μm particles was observed by MFI after 4 cycles of freezing and thawing. [Table 25-1] [Table 25-2] [Table 26-1] [Table 26-2] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31]

[0150] Stability studies have been conducted to evaluate the storage and accelerated stability of the liquid mAb1 drug product. The liquid drug product used in the storage and accelerated stability studies was manufactured by filling 2.5 mL of formulated drug substance into 6R ISO type 1 glass vials. The liquid drug product was incubated under storage, accelerated, and stress conditions. No obvious changes in the physical or chemical stability of the mAb1 liquid drug product were detected when stored at 5°C for up to 12 or 24 months (see Tables 32 and 33). These results indicate that the mAb1 liquid drug product is stable for at least 12 or 24 months at storage conditions. Results from the exploratory accelerated stability study are presented in Tables 34 and 35. A decrease in Region 1 with a concomitant increase in Region 2 was observed by CEX-UPLC when stored at 25°C / 60%RH for up to 6 months due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. An increase in region 1 with a concomitant decrease in region 2 was observed by iCIEF after incubation for up to 6 months at 25°C / 60% RH. No obvious changes were observed in other monitored attributes. Results from the exploratory stress stability study are presented in Tables 34-37. The mAb1 liquid drug product was physically and chemically stable when agitated (vortexed) for 120 minutes. An increase in HMW and LMW species was observed by SE-UPLC after 3 months of incubation at 40°C / 75% RH. An increase in LMW species was observed by reduced and non-reduced MCE after 3 months of incubation at 40°C / 75% RH. Different trends in charge variants were observed by CEX-UPLC and iCIEF due to different sensitivity of each assay. An increase in region 1 with a concomitant decrease in region 2 was observed by iCIEF after up to 3 months of incubation at 40°C / 75% RH, likely due to deamidation on asparagine or glutamine. After up to 2 months of incubation at 40°C / 75% RH, there is a decrease in region 1 with a concomitant increase in region 2 observed by CEX-UPLC. This is followed by a clear increase in region 1 with a concomitant decrease in region 2 observed by CEX-UPLC at 3 months, likely due to competing deamidation reactions.A decrease in region 3 was observed by iCIEF at 3 months when incubated at 40°C / 75%RH. An increase in 2-10um particles was observed by MFI after 4 cycles of freezing and thawing. Results from accelerated and stressed conditions indicated that HMW, LMW, and charge variants were the major degradation pathways for mAb1 liquid drug product. [Table 32-1] [Table 32-2] [Table 33-1] [Table 33-2] [Table 34-1] [Table 34-2] [Table 35-1] [Table 35-2] [Table 36-1] [Table 36-2] [Table 37-1] [Table 37-2]

[0151] Example 3: Freeze-drying cycle development The lyophilization process developed for clinical production consisted of freezing, primary drying, and secondary drying. The lyophilization process was developed using an FTS LyoStar™ III lyophilizer based on a partial cake collapse temperature of −31.2° C. determined for the frozen formulated drug substance using a lyophilization microscope. During primary drying, the product temperature did not exceed the partial cake collapse temperature, thereby maintaining cake integrity during the lyophilization cycle. The secondary drying process was developed to ensure that the drug product had a low residual moisture content.

[0152] The lyophilization cycle takes approximately 63 hours to produce lyophilized mAb1 drug product in 5 mL Type 1 glass vials containing 2.5 mL of 2 mg / mL mAb1 formulated drug substance. The lyophilization cycle includes the steps shown in Table 38 below. [Table 38]

[0153] Example 4: Development of a stable liquid anti-MUC16 x anti-CD3 bispecific antibody formulation for intravenous administration and stability of the formulation Development of intravenous (IV) formulations was performed, including formulations containing 5 mg / ml mAb1 and 50 mg / ml mAb1, to identify pH to minimize changes in glycosylation at HCDR3-Lys98 in the MUC16 binding arm, appropriate buffers and concentrations to maintain pH during manufacturing and overcome the observed Donnan effect, suitable thermal stabilizers at concentrations to maintain desired viscosity and tonicity, and suitable surfactants at sufficient concentrations for dilution during IV administration, all while maintaining a stable liquid formulation.

[0154] As discussed above, glycosylation at HC-CDR3-Lys98 of the MUC16 binding arm directly impacts potency. Deglycosylation leads to increased potency as measured by bioassay. The main formulation factor that affects the rate of deglycosylation in mAb1 is pH. The effect of pH on the levels of glycosylation and high molecular weight (HMW) species was investigated in liquid formulations by incubating 2 mg / mL mAb1 in 10 mM histidine, 10% (w / v) sucrose, and 0.05% (w / v) polysorbate 20 at three different pHs: 5.0, 5.5, and 6.0 for 36 months at 5 °C or 2 months at 25 °C (Figures 2A-2D). The major degradation pathways observed under these conditions are the formation of HMW species and deglycosylation at HC-CDR3-Lys98 as detected by CEX-UPLC. As the pH of the mAb1 formulations was reduced, the levels of HMW species formed and the rate of deglycosylation decreased.

[0155] Sodium acetate was selected as the buffer for mAb1 liquid formulations because histidine is not a good buffer at pH 5.0. The effect of pH was investigated by incubating 50 mg / mL mAb1 in 10 mM acetate with 5% (w / v) sucrose and five different pHs: 4.8, 5.0, 5.2, 5.5, and 5.7 under heat stress. After 28 days of incubation at 40°C, the major degradation pathways were the formation of HMW species and deglycosylation at HC-CDR3-Lys98. Consistent with the observations in histidine buffer, the level of HMW species formed and the rate of deglycosylation decreased at lower pH (Figures 3A-3B). pH 5.0 was selected as the target to produce a robust formulation where normal manufacturing variability would remain within the range required to stabilize mAb1.

[0156] The effect of acetate concentration on the stability of 150 mg / mL mAb1 formulations at pH 5.0 was examined in liquid formulations. Formulations containing acetate buffers ranging from 21 to 40 mM were incubated at 45°C for 14 days. The higher temperature allows for the rapid detection of protein degradation. Analysis revealed that the formation of HMW species and charge variants were the major degradation pathways. An increase in acetate concentration leads to an increase in HMW species, and 30 mM was selected based on the data. A concentration of 30 mM acetate can address the Donnan effect observed during the manufacturing process. As a result of the Donnan effect, 30 mM acetate is required to maintain the pH of the formulation at 5.0. This acetate concentration provided improved stabilization with respect to the formation of HMW species under this stress condition compared to formulations containing higher concentrations of acetate (Table 39). Therefore, 30 mM was selected as the acetate concentration for mAb1 liquid formulations for IV administration. [Table 39]

[0157] The effect of sucrose on the stability of mAb1 was examined in liquid formulations (containing 150 mg / mL mAb1, 30 mM acetate at pH 5.0, 0-13% [w / v] sucrose) under freeze / thaw and heat stress. Higher temperatures allow for rapid detection of protein degradation under these stresses. Under freeze / thaw stress, the formation of HMW species was the major degradation pathway (Table 40). Under heat stress at 45°C, the formation of HMW and LMW species and changes in charge variants were the major degradation pathways. Formulations containing higher concentrations of sucrose (≥10%) provided improved stabilization by reducing the levels of HMW and LMW species and the rate of deglycosylation at HC-CDR3-Lys98 (Table 41). To maintain the desired viscosity and tonicity of mAb1, 10% (w / v) sucrose was selected as the thermal stabilizer for the liquid formulation for IV administration. [Table 40] [Table 41]

[0158] The need for a surfactant was demonstrated during the initial development of the mAb1 formulation. When no surfactant was present, an increase in mAb1 HMW species was observed when the formulation was agitated by vortexing. The addition of a surfactant stabilized mAb1 against agitation stress. In the initial development, polysorbate 20 was selected as the surfactant due to its improved thermal stability compared to polysorbate 80. mAb1 formulations intended for IV administration also require polysorbate 20 in the formulation to stabilize mAb1 when diluted in 0.9% sodium chloride for IV administration.

[0159] The effect of polysorbate 20 on the stability of mAb1 was examined in a liquid formulation (50 mg / mL mAb1, 30 mM acetate, 10% [w / v] sucrose, 0–0.25% [w / v] polysorbate 20, pH 5.0) under agitation and heat stress.

[0160] Under agitation stress, mAb1 was stable in all liquid formulations tested (Table 42). Under heat stress at 45° C., the formation of HMW species and changes in charge variants were the major degradation pathways (Table 43). Increasing polysorbate 20 concentrations did not significantly affect the formation of charge variants, but some increase in the levels of HMW species formed was observed with increasing polysorbate 20 concentrations. As these mAb1 formulations are intended for IV delivery by dilution with 0.9% sodium chloride, the formulations will contain 0.2% (w / v) polysorbate 20. 0.2% polysorbate 20 stabilizes mAb1 and provides sufficient stabilization of mAb1 when diluted for IV administration. [Table 42-1] [Table 42-2] [Table 43-1] [Table 43-2]

[0161] Long-term accelerated stress stability studies were conducted to verify the stability of the liquid formulations. The results showed that these formulations provided adequate stability.

[0162] Example 5: Development of a stable liquid anti-MUC16 x anti-CD3 bispecific antibody formulation for subcutaneous administration and stability of the formulation Subcutaneous (SC) formulations were developed to identify excipients and concentrations optimized for SC administration and stability, including a formulation containing 150 mg / ml mAb1.

[0163] Buffer, pH, and surfactant parameters were identified for the IV formulation (Example 4) and these parameters were applied to the subcutaneous formulation. Surfactant (Polysorbate 20) concentration was also characterized during IV formulation development and the data was used to select the appropriate surfactant concentration for the subcutaneous formulation. Buffer, pH, and surfactant type and concentration were kept constant at the following levels during subcutaneous formulation development of mAb1: pH: 5.0 to minimize the rate of desaccharification and the formation of HMW species. Buffer: 30 mM sodium acetate to maintain pH 5.0 despite the Donnan effect observed during processing. Surfactant: 0.05% (w / v) polysorbate 20. To balance stability against stirring stress while minimizing the effect on thermal stability.

[0164] Other factors evaluated for the mAb1 SC formulation were: ●mAb1 concentration: Evaluated within the range of 50-150mg / mL A concentration of mAb1 to achieve the required clinical dose while maintaining stability for at least 24 months when stored at 2-8°C and a target osmolality of 290-400 mOsm / kg, and a viscosity of less than 20 cP at 20°C. Sucrose concentration: evaluated within the range of 2-10% A sucrose concentration to provide sufficient heat stability while minimizing the impact on osmolality and viscosity. At least 2% (w / v) sucrose is required to stabilize mAb1 against freeze / thaw stress Arginine concentration: Evaluated within the range of 0 to 100 mM Evaluate arginine's ability to reduce viscosity and evaluate its effect on stability and osmolality

[0165] The formulations evaluated for SC administration are shown in Table 44 below. [Table 44]

[0166] Viscosity Analysis - Figure 4 shows the dependence of mAb1 viscosity as a function of mAb1 concentration, sucrose concentration, and arginine concentration. The following observations were made from this study: (i) the major factor contributing to viscosity is the mAb1 concentration. Over the range of 50-150 mg / mL, the viscosity increases exponentially from about 2 cP to about 11 cP (at 20°C). (ii) there is a slight dependence of viscosity on sucrose. At a mAb1 concentration of 150 mg / mL, the viscosity increases from about 11 cP to 13 cP (at 20°C) when sucrose is varied from 2 to 10% (w / v). (iii) the effect of arginine concentration on viscosity is similar in magnitude to sucrose, but increasing arginine decreases the viscosity from about 11 cP to 9 cP (at 20°C).

[0167] Osmolality Analysis - Figure 5 shows the dependence of mAb1 osmolality as a function of mAb1 concentration, sucrose concentration, and arginine concentration. The following observations were made from this study: (i) mAb1 concentration makes a negligible contribution to osmolality over the range of 50-150 mg / mL. (ii) The major factor contributing to osmolality is sucrose. At a mAb1 concentration of 150 mg / mL, the osmolality increases from about 90 mOsm / kg to about 410 mOsm / kg when sucrose is varied from 2 to 10% (w / v). (iii) An increase in arginine concentration also results in an increase in osmolality. At mAb1 at 150 mg / mL, an increase in arginine from 0 to 100 mM increases the osmolality from about 90 mOsm / kg to about 300 mOsm / kg.

[0168] Stability Analysis - Figure 6 shows the stability of mAb1 as a function of mAb1 concentration, sucrose concentration, and arginine concentration. In this study, HMW species formation at 25°C / 60% RH and 40°C / 75% RH was evaluated. In addition, the loss of acidic species and the loss of glycated species were evaluated under the same conditions. The following observations were made: (i) increasing sucrose concentration led to a decrease in the rate of HMW formation at both 25°C / 60% RH and 40°C / 75% RH. (ii) increasing arginine concentration led to a decrease in the rate of HMW formation at 25°C / 60% RH but an increase in the rate of HMW formation at 40°C / 75% RH. (iii) increasing sucrose concentration led to a reduction in the rate of acidic and glycated species formation at 25°C / 60% RH. (A less negative rate means less degradation over time, since acidic and glycated species decrease over time) (iv) Increasing arginine concentration led to a decreased rate of glycated species formation at 25°C / 60% RH.

[0169] Based on previous development studies (e.g., Examples 1 and 2) and this study, two lead formulations were selected for further stability, viscosity, and osmolality evaluation. The two lead formulations and their respective viscosities and osmolality are shown in Table 45 below. Both formulations met the viscosity and osmolality targets at a mAb1 concentration of 150 mg / mL. Both formulations showed comparable stability when incubated at 40° C. / 75% RH for 3 months, 25° C. / 60% RH for 6 months, or 2-8° C. for 6 months (FIGS. 7A and 7B). Both formulations showed comparable changes in HMW and glycated species. [Table 45]

[0170] Based on these data, both formulations are comparable. Arginine was not selected as an excipient in the subcutaneous formulation because it provides little improvement in stability or viscosity, but results in an increase in osmolality. Thus, a preferred exemplary subcutaneous formulation is 150 mg / mL mAb1, 30 mM acetate, pH 5.0, 8% w / v sucrose, and 0.05% w / v polysorbate 20.

[0171] Stability studies were also initiated to evaluate the storage, stress, and accelerated stability of the mAb1 formulation. The stability studies included a subcutaneous (SC) formulation (150 mg / mL mAb1 in 30 mM sodium acetate, 8% (w / v) sucrose, 0.05% (w / v) polysorbate 20, pH 5.0) stored in Schott 6R borosilicate glass vials. The liquid formulation was incubated under storage, stress, and accelerated conditions. The stress and accelerated conditions were chosen to simulate conditions beyond those that a drug product would experience during manufacturing and handling, and to elucidate degradation pathways of mAb1. Stability under additional stresses including agitation and freeze / thaw was also evaluated.

[0172] The antibody in the formulation evaluated (mAb1) was physically and chemically stable when stored at 5° C. for at least 6 months (Table 46). No obvious changes in stability were observed in any of the monitored attributes at 5° C. in 6R vials. Results from analysis of mAb1 formulations after incubation under accelerated and stress conditions are provided in Table 47. After 6 months of incubation at 25° C. / 60% RH, an increase of 1.7% HMW species was observed by SE-UPLC. Different trends in charge variants were observed by CEX-UPLC and iCIEF due to different sensitivity of each assay. An increase in region 1 with a concomitant decrease in region 2 was observed by iCIEF. A decrease in region 1 with a concomitant increase in region 2 was observed by CEX-UPLC when stored at 25° C. / 60% RH for up to 6 months due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm (deglycosylation also occurs under heat stress conditions). Results from the accelerated conditions showed that the liquid formulation was stable under heat stress in 6R vials.

[0173] After 3 months of incubation at 40°C / 75% RH, a 15.9 and 1.1% increase in HMW and LMW species, respectively, was observed by SE-UPLC. Different trends in charge variants were observed by CEX-UPLC and iCIEF, due to different sensitivities of each assay. An increase in region 1 with a concomitant decrease in regions 2 and 3 was observed by iCIEF after up to 3 months of incubation at 40°C / 75% RH, likely due to deamidation on asparagine or glutamine. After up to 2 months of incubation at 40°C / 75% RH, there is a decrease in region 1 with a concomitant increase in region 2 observed by CEX-UPLC due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm (deglycosylation also occurs in heat stress conditions). This is followed by a clear increase in region 1 with a concomitant decrease in region 2 observed by CEX-UPLC at 3 months, likely due to competing deamidation reactions. There was also a 16.3% increase in region 3 after 3 months at 40°C / 75%RH observed by CEX-UPLC. This increase was determined to be composed primarily of oligomeric species of mAb1, including tetrameric, pentameric, hexameric, and heptameric species. Results from incubation of mAb1 formulations at 40°C / 75%RH indicated that the formation of HMW and LMW species, as well as changes in the distribution of charge variants, were the major degradation pathways for the mAb1 drug product.

[0174] The mAb1 formulation was physically and chemically stable when vortexed for either 60 or 120 minutes (Table 48). No obvious changes in physical or chemical stability were detected in any of the attributes monitored. The mAb1 formulation was also physically and chemically stable when subjected to four freeze and thaw cycles (Table 48). No obvious changes in physical or chemical stability were detected in any of the attributes monitored. [Table 46-1] [Table 46-2] [Table 47-1] [Table 47-2] [Table 47-3] [Table 48-1] [Table 48-2]

[0175] Additional stability studies were initiated to determine the long-term storage, accelerated (temperature above storage conditions), and stress stability (40°C / 75% RH, agitation, freeze and thaw) of the mAb1 formulation at 150 mg / mL antibody. The mAb1 formulation containing 150 mg / mL antibody was filled into 5 mL polycarbonate vials for agitation, freeze / thaw, frozen storage, and accelerated and stress storage conditions. The polycarbonate vials are representative of the storage containers used for mAb1 formulations (formulated drug substance) manufactured in a GMP facility. The formulations tested contained 150 mg / mL purified mAb1 in an aqueous buffer solution containing 30 mM sodium acetate, 8% (w / v) sucrose, and 0.05% (w / v) PS20 at pH 5.0.

[0176] No obvious changes in the physical or chemical stability of mAb1 formulations were detected when stored at -80°C and -30°C for up to 6 months (Tables 49 and 50). These results indicate that mAb1 (150 mg / mL) is stable for at least 6 months when stored frozen at storage conditions.

[0177] Results from the exploratory accelerated stability study are presented in Tables 51A and 51B. No obvious changes in monitored attributes were observed after incubation of mAb1 formulations (150 mg / mL antibody) at -20°C for up to 6 months. An increase in protein concentration was observed by SoloVPE after 6 months of incubation at 25°C / 60%RH, likely due to sample evaporation. An increase in HMW species was observed by SE-UPLC after 6 months of incubation at 5°C and 25°C / 60%RH. An increase in region 1 (acidic species) with a concomitant decrease in region 2 (major peak) was observed by CEX-UPLC after 6 months of incubation at 25°C / 60%RH due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. An increase in region 1 (acidic species) with a concomitant decrease in region 2 (major peak) was observed by iCIEF after 6 months of incubation at 25°C / 60%RH, likely due to deamidation. These results indicate that mAb1 (150 mg / mL) formulations can withstand incubation at -20°C for at least 6 months and at 5°C for 3 months without compromising either the physical or chemical stability of the protein. mAb1 formulations can also withstand short-term exposure to temperatures of 25°C / 60%RH.

[0178] Results from the exploratory stress stability study are presented in Tables 51A and 51B, and Table 52. mAb1 (150 mg / mL) formulations were physically and chemically stable when agitated (vortexed) for up to 120 minutes or subjected to up to 4 cycles of freezing and thawing. An increase in protein concentration was observed by SoloVPE after up to 3 months of incubation at 40° C. / 75% RH, likely due to sample evaporation. An increase in HMW and LMW species was observed by SE-UPLC after up to 3 months of incubation at 40° C. / 75% RH. A decrease in region 1 with a concomitant increase in region 2 was observed by CEX-UPLC after incubation at 40° C. / 75% RH due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm. After 2 months of incubation at 40° C. / 75% RH, there is a decrease in region 1 and a concomitant increase in region 2. However, after the 3 month time point, the trend was reversed and there was a clear increase in regions 1 and 3 with a concomitant decrease in region 2. The increase in region 1 was likely due to competitive deamidation on asparagine or glutamine, while the increase in region 3 was determined to be composed primarily of oligomeric species of mAb1, including tetrameric, pentameric, hexameric, and heptameric species. Results from accelerated and stressed conditions indicated that HMW, LMW, and charge variants were the major degradation pathways for mAb1 (150 mg / mL). [Table 49-1] [Table 49-2] [Table 50-1] [Table 50-2] [Table 51-1] [Table 51-2] [Table 52-1] [Table 52-2] [Table 53-1] [Table 53-2]

[0179] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. *******

Claims

1. 1. A stable liquid pharmaceutical formulation comprising: (a) a bispecific antibody at a concentration of 1 mg / ml ± 0.1 mg / ml to 200 mg / ml ± 20 mg / ml, the bispecific antibody comprising a first antigen-binding domain that specifically binds to human MUC16 and a second antigen-binding domain that specifically binds to human CD3, the first antigen-binding domain comprising three heavy chain complementarity determining regions (CDRs) (A1-HCDR1, A1-HCDR2, and A1-HCDR3) comprised in a heavy chain variable region (HCVR) and three light chains (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR); a bispecific antibody comprising three heavy chain CDRs (A1-HCDR1, A2-HCDR2, and A2-HCDR3) comprised in a light chain variable region (LCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised in a light chain variable region (LCVR), wherein A1-HCDR1, A1-HCDR2, and A1-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 7, 8, and 9, respectively, A2-HCDR1, A2-HCDR2, and A2-HCDR3 comprise the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively, and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (b) a buffer containing acetate at a concentration of 10 mM±1 mM to 50 mM±5 mM; (c) an organic co-solvent comprising polysorbate at a concentration of 0.01%±0.005% to 0.5%±0.05% w / v; (d) a stabilizer comprising sucrose at a concentration of 5%±1% to 20%±4% w / v; Including, A pharmaceutical formulation, wherein the formulation has a pH of 5.0±0.

5.

2. (a) the antibody concentration is from 5 mg / ml ± 0.5 mg / ml to 50 mg / ml ± 5 mg / ml, 5 mg / ml ± 0.5 mg / ml, or 50 mg / ml ± 5 mg / ml; (b) the antibody concentration is between 125 mg / ml and 175 mg / ml, or 150 mg / ml ± 10 mg / ml; (c) the acetate buffer concentration is 25 mM ± 2.5 mM to 35 mM ± 3.5 mM, or 30 mM ± 3 mM; (d) the polysorbate concentration is 0.1%±0.05% to 0.3%±0.03% w / v, or 0.2%±0.02% w / v; (e) the polysorbate concentration is between 0.01% w / v and 0.1% w / v, or 0.05% w / v±0.01% w / v; (f) the polysorbate is polysorbate 20; (g) the sucrose concentration is between 7%±0.5% and 12%±0.5% w / v, or 10%±1% w / v; and / or (h) the sucrose concentration is between 4% and 12% w / v, or 8% w / v±1% w / v; 2. The pharmaceutical formulation of claim 1.

3. 2. The pharmaceutical formulation of claim 1, (A) (a) 5 mg / ml ± 0.5 mg / ml of an antibody; (b) 25 mM±2 mM to 35 mM±2 mM acetate buffer; (c) 0.1%±0.05% to 0.3%±0.05% w / v polysorbate; and (d) 5%±1% to 15%±3% w / v sucrose; at pH 5.0±0.5, Optionally, the pharmaceutical formulation comprises: (i) 5 mg / ml ± 0.5 mg / ml of an antibody; (ii) 30 mM ± 1 mM acetate buffer; (iii) 0.2%±0.02% w / v polysorbate 20; and (iv) 10%±1% w / v sucrose; and at pH 5.0±0.3, or (B) (a) 50 mg / ml ± 5 mg / ml of an antibody; (b) 25 mM±2 mM to 35 mM±2 mM acetate buffer; (c) 0.1%±0.05% to 0.3%±0.05% w / v polysorbate; and (d) 5%±1% to 15%±3% w / v sucrose; at pH 5.0±0.5, Optionally, the pharmaceutical formulation comprises: (i) 50 mg / ml ± 0.5 mg / ml of an antibody; (ii) 30 mM ± 1 mM acetate buffer; (iii) 0.2%±0.02% w / v polysorbate 20; and (iv) 10%±1% w / v sucrose; and 2. The pharmaceutical formulation of claim 1, comprising:

4. The formulation comprises: (a) contains 2.5% or less high molecular weight (HMW) species after 12 or 24 months storage at 5° C. as determined by SE-UPLC; (b) contains 3.5% or less high molecular weight (HMW) species after 6 months storage at 25° C. and 60% relative humidity as determined by SE-UPLC; (c) contains 1.5% or less high molecular weight (HMW) species after 12 months storage at −30° C., or 2.0% or less HMW species after 24 months storage at −30° C., as determined by SE-UPLC; or (d) contains less than 1.5% high molecular weight (HMW) species after 12 months storage at −80° C., or less than 2.0% HMW species after 24 months storage at −30° C., as determined by SE-UPLC; The pharmaceutical formulation according to any one of claims 1 to 3.

5. 5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the formulation contains 40% or less glycosylated species variants, the glycosylated species variants comprising a glycosylation at residue 2 of A1-HCDR3 set forth in SEQ ID NO:9, and optionally, the formulation comprises 5% or less change in glycosylated species after 6 months storage at 5°C, the glycosylated species comprising a glycosylation at residue 2 of A1-HCDR3 set forth in SEQ ID NO:9, as determined by cation exchange ultra performance liquid chromatography (CEX-UPLC) or by liquid chromatography mass spectrometry (LC-MS).

6. The first antigen-binding domain comprises: (a) a HCVR having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID NO: 6, wherein the second antigen-binding domain comprises a HCVR having at least 90% identity to the amino acid sequence of SEQ ID NO: 5 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID NO: 6; and / or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 4 and an LCVR comprising the amino acid sequence of SEQ ID NO: 6, wherein the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 5 and an LCVR comprising the amino acid sequence of SEQ ID NO: 6; The pharmaceutical formulation according to any one of claims 1 to 5.

7. A stable pharmaceutical formulation comprising: (a) (i) a bispecific antibody at 5 mg / ml ± 0.5 mg / ml comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a HCVR comprising an amino acid of SEQ ID NO: 4 and a LCVR comprising an amino acid of SEQ ID NO: 6, and the second antigen-binding domain comprises a HCVR comprising an amino acid of SEQ ID NO: 5 and a LCVR comprising an amino acid of SEQ ID NO: 6; (ii) 30 mM ± 1 mM sodium acetate buffer, pH 5.0 ± 0.2; (iii) 0.2%±0.02% w / v polysorbate 20; and (iv) 10%±1% w / v sucrose; (b) (i) a bispecific antibody at 50 mg / ml ± 5 mg / ml comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 4 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 5 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6; (ii) 30 mM ± 1 mM sodium acetate buffer, pH 5.0 ± 0.2; (iii) 0.2%±0.02% w / v polysorbate 20; and (iv) 10%±1% w / v sucrose; or (c) (i) a bispecific antibody at 150 mg / ml ± 15 mg / ml comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a HCVR comprising an amino acid of SEQ ID NO: 4 and a LCVR comprising an amino acid of SEQ ID NO: 6, and the second antigen-binding domain comprises a HCVR comprising an amino acid of SEQ ID NO: 5 and a LCVR comprising an amino acid of SEQ ID NO: 6; (ii) 30 mM ± 1 mM sodium acetate buffer, pH 5.0 ± 0.2; (iii) 0.05%±0.01% w / v of polysorbate 20; and (iv) 8%±1% w / v sucrose; Pharmaceutical preparations.

8. 8. The pharmaceutical formulation of any one of claims 1 to 7, wherein the formulation contains less than or equal to 5% change in glycosylated species after 6 months of storage at 5°C, and the glycosylated species contains glycosylation at residue 98 of HCVR set forth in SEQ ID NO: 4 as determined by cation exchange ultra performance liquid chromatography (CEX-UPLC) or by liquid chromatography mass spectrometry (LC-MS).

9. 9. The pharmaceutical formulation of any one of claims 1 to 8, wherein the antibody comprises a human IgG heavy chain constant region attached to the HCVR of each of the first and second antigen binding domains, respectively, optionally wherein the heavy chain constant region is of isotype IgG1 or isotype IgG4.

10. (a) the heavy chain constant region attached to the HCVR of the first antigen-binding domain or the heavy chain constant region attached to the HCVR of the second antigen-binding domain, but not both, comprise amino acid modifications that reduce Protein A binding relative to a heavy chain of the same isotype lacking the modification, optionally wherein the modifications comprise H435R and Y436F substitutions (EU numbering) in heavy chains of isotype IgG1 or IgG4; or (b) the antibody comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; In some cases, (i) the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 16 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 17; or (ii) the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 19; 10. The pharmaceutical formulation of claim 9.

11. the antibody comprises a first heavy chain comprising the HCVR of the first antigen-binding domain and a second heavy chain comprising the HCVR of the second antigen-binding domain, the first heavy chain comprising residues 1-442 of the amino acid sequence of SEQ ID NO:1 and the second heavy chain comprising residues 1-449 of the amino acid sequence of SEQ ID NO:2; 11. The pharmaceutical formulation of any one of claims 1 to 10, optionally wherein the antibody comprises a common light chain comprising the LCVR of the first and second antigen binding domains, and wherein the common light chain comprises the amino acid sequence of SEQ ID NO:

3.

12. 8. The pharmaceutical formulation of claim 7, wherein the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 4 and an LCVR comprising the amino acid sequence of SEQ ID NO: 6, and the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 5 and an LCVR comprising the amino acid sequence of SEQ ID NO:

6.

13. A product comprising: (a) a pharmaceutical composition, the composition comprising the pharmaceutical formulation according to any one of claims 1 to 12, the composition being contained in a container; (b) (i) a container containing a composition comprising the pharmaceutical formulation of any one of claims 1 to 12, and instructions for use of said composition; or (c) a container containing a composition comprising the pharmaceutical formulation according to any one of claims 1 to 12. A product that is.

14. The container comprises: (a) a vial or a 2 ml, 5 ml, or 10 ml Type 1 clear glass vial; (b) a syringe, or a low tungsten glass syringe; (c) a pre-filled syringe; or (d) Self-injector 14. The product of claim 13,

15. 13. A unit dosage form comprising the pharmaceutical formulation of any one of claims 1 to 12, wherein the antibody is present in an amount of 0.1 mg to 500 mg, 1 to 20 mg, or 100 to 200 mg, and the unit dosage form is a glass vial, a pre-filled syringe, or an autoinjector.

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