Pharmaceutical compositions and methods of use of mosunetuzumab

Abstract

To provide pharmaceutical formulations which are stable and protected from loss, e.g., due to surface adsorption, and methods for treating or delaying progression of a cancer.SOLUTION: A pharmaceutical composition comprises mosunetuzumab, polysorbate 20 (PS20), methionine, a buffering agent, and a carrier, wherein the concentration of PS20 is from 0.01% to 0.1% weight-by-volume (w / v), the concentration of rnethionine is from 1 mM to 50 mM, and the concentration of the buffering agent is from 5 mM to 20 mM.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. Said XML copy, created on March 23, 2023, is named 50474-163WO2_Sequence_Listing_3_23_23 and is 33,967 bytes in size.

[0002] FIELD OF THE INVENTION The present disclosure relates to compositions (eg, pharmaceutical compositions) comprising mosunetuzumab and methods of use thereof. [Background technology]

[0003] background Mosunetuzumab is being used as a treatment for cancer, e.g., a CD20-positive cell proliferative disorder (e.g., a B-cell proliferative disorder, e.g., non-Hodgkin's lymphoma (NHL) (e.g., follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), etc.) or B-cell chronic lymphocytic leukemia (CLL). Mosunetuzumab may be formulated in an aqueous carrier for administration to a subject, e.g., by intravenous administration.

[0004] One of the major challenges in the development of biotechnology therapeutics is protein stability, which must be maintained throughout multiple process steps before reaching market. Furthermore, protein stability must be maintained not only during storage but also during administration to patients. During the storage, handling, and administration of such pharmaceutical compositions, it is necessary to mitigate potential losses of mosunetuzumab due to degradation and surface adsorption, such as protein adsorption to the surfaces of filters, storage canisters, tubing, syringes, intravenous infusion bags, and other containers. When pharmaceutical compositions contain relatively low concentrations of mosunetuzumab, protein loss can be dramatically increased by these factors, potentially reducing the therapeutic efficacy of the pharmaceutical composition.

[0005] Therefore, there is a need in the art to develop pharmaceutical formulations in which mosunetuzumab is stable and protected from loss due to, for example, surface adsorption. Summary of the Invention

[0006] Disclosure Overview The present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising low concentrations of mosunetuzumab and methods of using the same. The disclosed compositions and related methods address the problem of delivering mosunetuzumab formulated at low concentrations, ensuring that patients receive the intended dose of mosunetuzumab with little or no protein loss during storage and administration.

[0007] In one aspect, the disclosure provides a pharmaceutical composition comprising mosunetuzumab, a surfactant (e.g., polysorbate 20 (PS20)), methionine, a buffering agent, and a carrier, wherein the surfactant has a concentration of 0.01% to 0.1% weight / volume (w / v), the methionine has a concentration of 1 mM to 50 mM, and the buffering agent has a concentration of 5 mM to 20 mM.

[0008] In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.01% to 0.1% weight / volume (e.g., 0.01% to 0.025%, 0.025% to 0.5%, 0.05% to 0.075%, or 0.075% to 0.1% (w / v), e.g., 0.01% to 0.02%, 0.02% to 0.03%, 0.03% to 0.04%, 0.04% to 0.05%, 0.05% to 0.06%, 0.06% to 0.07%, 0.07% to 0.08%, 0.08% to 0.09%, or 0.09% to 0.09%). 0.1% (w / v), e.g., about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, about 0.05%, about 0.055%, about 0.06%, about 0.065%, about 0.07%, about 0.075%, about 0.08%, about 0.085%, about 0.09%, about 0.095%, or about 0.1% (w / v). In certain embodiments, the concentration of the surfactant (e.g., PS20) is about 0.06 (w / v) (i.e., about 0.6 mg / ml).

[0009] In some embodiments, the concentration of methionine is 1 mM to 50 mM (e.g., 1 mM to 10 mM, 10 mM to 20 mM, 20 mM to 30 mM, 30 mM to 40 mM, or 40 mM to 50 mM, e.g., 5 mM to 45 mM, 10 mM to 40 mM, 15 mM to 35 mM, or 20 mM to 30 mM, e.g., 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, In some embodiments, the concentration of methionine is about 2.5 mM to about 20 mM. In a particular embodiment, the concentration of methionine is about 10 mM.

[0010] In some embodiments, the concentration of the buffering agent is 5 mM to 20 mM (e.g., 5 mM to 10 mM, 10 mM to 15 mM, or 15 mM to 20 mM, e.g., 6 mM to 18 mM, 7 mM to 16 mM, 8 mM to 15 mM, 9 mM to 12 mM, or 8 mM to 12 mM, e.g., 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 embodiments, the concentration of the buffering agent is about 8 mM to about 12 mM. In certain embodiments, the concentration of the buffering agent is about 10 mM.

[0011] In some embodiments, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is 100 or less, e.g., 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 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 or less, or 1 or less, e.g., 0.5 to 100, 0.5 to 50, 0.5 to 10, 0.5 to 5, 0.5 to 1, 1 to 5, 2 to 4, 5 to 100, 10 to 70, 10 to 50, 10 to 30, or 50 to 100. In certain embodiments, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is 1 to 100. In some embodiments, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is 50-100, 60-80, or 65-75, e.g., about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In certain embodiments, the molar ratio of surfactant to mosunetuzumab is about 71.

[0012] In some embodiments, the concentration of mosunetuzumab is about 0.5 mg / ml to about 2 mg / ml (e.g., about 0.5 to about 1.5 mg / ml, about 0.7 to about 1.3 mg / ml, about 0.8 to about 1.2 mg / ml, about 0.9 to about 1.1 mg / ml, about 0.5 to about 1.0 mg / ml, or about 1 to about 1.5 mg / ml; e.g., about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, or about 2.0 mg / ml). In certain embodiments, the concentration of mosunetuzumab is about 1 mg / ml. In some embodiments, the pharmaceutical composition is formulated as a drug formulation (DP).

[0013] In some embodiments, the pharmaceutical composition formulated as a DP has a mosunetuzumab concentration of about 1 mg / ml and / or a molar ratio of surfactant (e.g., PS20) to mosunetuzumab of 50-100, 60-80, or 65-75, e.g., about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is 65-75. In certain embodiments, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab in the DP is about 71.

[0014] In some embodiments, the pharmaceutical composition formulated as a DP has a mosunetuzumab concentration of about 1 mg / ml and / or a molar ratio of surfactant (e.g., P188) to mosunetuzumab of 5 to 50, 5 to 25, 10 to 15, or 15 to 20, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In a particular embodiment, the molar ratio of surfactant (e.g., P188) to mosunetuzumab is about 14. In another particular embodiment, the molar ratio of surfactant (e.g., P188) to mosunetuzumab is about 17.

[0015] In some embodiments, the buffer is histidine, phosphate, succinate, acetate, or a combination thereof. For example, in certain embodiments, the buffer is histidine, e.g., histidine acetate. The concentration of the buffer (e.g., histidine, e.g., histidine acetate) can be, for example, 8 mM to 12 mM, e.g., about 8 mM, about 9 mM, about 10 mM, about 11 mM, or about 12 mM. In certain embodiments, the concentration of the buffer (e.g., histidine, e.g., histidine acetate) is about 10 mM.

[0016] In some embodiments, the pharmaceutical composition further comprises a tonicity agent, such as a sugar, an amino acid, or a salt. In embodiments where the tonicity agent is a sugar, the sugar can be, for example, sucrose, glucose, glycerol, or trehalose. In certain embodiments, the sugar is sucrose. In some embodiments, the concentration of the tonicity agent (e.g., a sugar, e.g., sucrose) is between 100 mM and 500 mM (e.g., between 100 mM and 120 mM, 120 mM and 140 mM, 140 mM and 160 mM, 160 mM and 180 mM, 180 mM and 200 mM, 200 mM and 220 mM, 220 mM and 240 mM, 240 mM and 260 mM, 260 mM and 280 mM, 280 mM and 300 mM, 300 mM and 320 mM, 320 mM and 340 mM, 340 mM and 360 mM, 360 mM and 380 mM, 380 mM and 400 mM, 400 mM and 450 mM). The concentration is typically 100 mM to 420 mM, 420 mM to 440 mM, 440 mM to 460 mM, 460 mM to 480 mM, or 480 mM to 500 mM, for example, 100 mM to 400 mM, 150 mM to 350 mM, or 200 mM to 300 mM, for example, about 100 mM, about 150 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM). In certain embodiments, the concentration of the isotonic agent (eg, a sugar, such as sucrose) is about 240 mM.

[0017] In some embodiments, the pharmaceutical composition has a pH of 4.5 to 8 (e.g., 4.5 to 5.0, 5.0 to 5.5, 5.5 to 6.0, 6.0 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0, e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4 The pharmaceutical composition has a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the pharmaceutical composition has a pH of 5.5 to 6.1. In certain embodiments, the pharmaceutical composition has a pH of about 5.8.

[0018] In some embodiments, mosunetuzumab has a methionine at position 257 (i.e., Met257 or M257) (according to the EU index) of the Fc region. In some embodiments, oxidation of methionine at position 257 of the Fc region is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%; e.g., 0-9%, 0-8%, 0-7%, 0-6%, 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-10%, 2-9%, 3-8%, or 4-7%; e.g., about 10%, about 9%, about 8%, about 7%, about 6%, about 4%, about 4%, about 3%, about 2%, about 1%, or about 0%) over a 2-week period at 40°C. In some embodiments, the oxidation of methionine at position 257 of the Fc region is about 6% or less over two weeks at 40°C.

[0019] In another embodiment, the pharmaceutical composition comprises mosunetuzumab, a surfactant (e.g., PS20 or poloxamer 188 (P188)), methionine, and a carrier, wherein the pharmaceutical composition has a pH of about 5.8. In some embodiments, the concentration of mosunetuzumab is about 0.5 mg / ml to about 2 mg / ml (e.g., about 0.5 to about 1.5 mg / ml, about 0.7 to about 1.3 mg / ml, about 0.8 to about 1.2 mg / ml, about 0.9 to about 1.1 mg / ml, about 0.5 to about 1.0 mg / ml, or about 1 to about 1.5 mg / ml; e.g., about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, or about 2.0 mg / ml). In certain embodiments, the concentration of mosunetuzumab is about 1 mg / ml.

[0020] In some embodiments, the concentration of the surfactant is 0.05% to 0.1% (w / v) (e.g., 0.05% to 0.075% or 0.075% to 0.1% (w / v), e.g., 0.05% to 0.06%, 0.06% to 0.07%, 0.07% to 0.08%, 0.08% to 0.09%, or 0.09% to 0.1% (w / v), e.g., about 0.05%, about 0.055%, about 0.06%, about 0.065%, about 0.07%, about 0.075%, about 0.08%, about 0.085%, about 0.09%, about 0.095%, or about 0.1% (w / v)), and the concentration of methionine is about 10 mM.

[0021] In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to mosunetuzumab is less than or equal to 100. In some embodiments, the surfactant is PS20 and the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is less than or equal to 100, e.g., less than or equal to 90, less than or equal to 80, less than or equal to 70, less than or equal to 60, less than or equal to 50, less than or equal to 40, less than or equal to 30, less than or equal to 20, less than or equal to 10, less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1, e.g., 0.5 to 100, 0.5 to 50, 0.5 to 10, 0.5 to 5, 0.5 to 1, 1 to 5, 2 to 4, 5 to 100, 10 to 70, 10 to 50, 10 to 30, 15 to 20, 50 to 100, 60 to 80, or 65 to 75. In certain embodiments, the molar ratio of PS20 to mosunetuzumab is 50-100. In some embodiments, the molar ratio of PS20 to mosunetuzumab is 50-100, 60-80, or 65-75, e.g., about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, the surfactant is PS20, and the concentration of PS20 is about 0.06% (w / v). In certain embodiments, the molar ratio of PS20 to mosunetuzumab is about 71.

[0022] In some embodiments, the concentration of mosunetuzumab is about 0.5 mg / ml to about 2 mg / ml, e.g., about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, or about 2.0 mg / ml. In certain embodiments, the concentration of mosunetuzumab is about 1 mg / ml. In some embodiments, the pharmaceutical composition is formulated as a DP.

[0023] In some embodiments, the molar ratio of surfactant (e.g., P188) to mosunetuzumab is less than or equal to 100. In some embodiments, the concentration of P188 is about 0.08% (w / v) or about 0.1% (w / v). In some embodiments, the molar ratio of P188 to mosunetuzumab is 100 or less, e.g., 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 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 or less, or 1 or less, e.g., 0.5 to 100, 0.5 to 50, 0.5 to 10, 0.5 to 5, 0.5 to 1, 1 to 5, 2 to 4, 1 to 20, 1 to 30, 1 to 15, 10 to 20, 5 to 100, 10 to 70, 5 to 50, 10 to 50, 10 to 30, 15 to 20, or 10 to 15. In some embodiments, the molar ratio of P188 to mosunetuzumab is 5 to 50. In some embodiments, the molar ratio of P188 to mosunetuzumab is 5 to 50, 5 to 25, 10 to 15, or 15 to 20, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In a particular embodiment, the molar ratio of P188 to mosunetuzumab is about 14. In another particular embodiment, the molar ratio of P188 to mosunetuzumab is about 17.

[0024] In some embodiments, the pharmaceutical composition further comprises histidine acetate at a concentration of about 10 mM and / or sucrose at a concentration of about 240 mM.

[0025] In another aspect, the disclosure features a pharmaceutical composition including mosunetuzumab, PS20, and a carrier, wherein the molar ratio of PS20 to mosunetuzumab is less than or equal to 100, and the concentration of PS20 is between 0.01% weight / volume and 0.1% weight / volume (w / v).

[0026] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., a liquid formulation for infusion, a liquid formulation for injection, a liquid formulation for dilution, etc.). In certain embodiments, the pharmaceutical composition is a liquid formulation for dilution. In certain embodiments, the liquid formulation for dilution is supplied in a container having a volume of about 50 ml (e.g., about 40 ml, about 45 ml, about 46 ml, about 47 ml, about 48 ml, about 49 ml, about 50 ml, about 51 ml, about 52 ml, about 53 ml, about 54 ml, about 55 ml, or about 60 ml). In some embodiments, the volume of the liquid formulation for dilution is 20 to 40 ml (e.g., 20 to 30 ml, 30 to 40 ml, 20 to 35 ml, 25 to 40 ml, 25 to 35 ml, or 28 to 32 ml; e.g., about 20 ml, about 25 ml, about 26 ml, about 27 ml, about 28 ml, about 29 ml, about 30 ml, about 31 ml, about 32 ml, about 33 ml, about 34 ml, about 35 ml, or about 40 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 30 ml. In another specific embodiment, the liquid formulation for dilution is supplied in a container having a volume of about 2 ml (e.g., about 1 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, about 2 ml, about 2.1 ml, about 2.2 ml, about 2.3 ml, about 2.4 ml, about 2.5 ml, or about 3 ml). In some embodiments, the volume of the liquid formulation for dilution is 0.2 to 2 ml (e.g., 0.2 to 1.5 ml, 0.5 to 2 ml, 0.5 to 1 ml, or 0.8 to 1.2 ml; e.g., about 0.2 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 1.1 ml, about 1.2 ml, about 1.3 ml, about 1.4 ml, about 1.5 ml, or about 2 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 1 ml.

[0027] In some embodiments, the liquid formulation is for dilution with a diluent. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the saline comprises sodium chloride (NaCl). In some embodiments, the saline comprises 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl.

[0028] In some embodiments, the pharmaceutical composition is present in a container such as a tank (e.g., a mini-tank) (e.g., a stainless steel container or a nickel alloy container (e.g., HASTELLOY®)), or a can (e.g., a mini-can).

[0029] In some embodiments, the pharmaceutical composition contains 1,000 or fewer particles per ml having a diameter of 2 μm or more (e.g., 900 or fewer, 800 or fewer, 700 or fewer, 600 or fewer, 500 or fewer, 400 or fewer, 300 or fewer, 200 or fewer, or 100 or fewer particles per ml having a diameter of 2 μm or more, e.g., 0 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1,000 or fewer particles per ml having a diameter of 2 μm or more). In some embodiments, the carrier is water.

[0030] In some embodiments, the pharmaceutical composition has a shelf life of at least 36 months (e.g., at least 38 months, at least 40 months, at least 42 months, at least 44 months, at least 46 months, at least 48 months, at least 60 months, at least 72 months, or at least 96 months) when stored at 5°C ± 3°C and protected from light. In some embodiments, the pharmaceutical composition is stable through one or more freeze-thaw cycles (e.g., two or more freeze-thaw cycles, three or more freeze-thaw cycles, four or more freeze-thaw cycles, five or more freeze-thaw cycles, six or more freeze-thaw cycles, eight or more freeze-thaw cycles, or more freeze-thaw cycles). In certain embodiments, the pharmaceutical composition is stable through three or more freeze-thaw cycles. In some embodiments, the pharmaceutical composition is stable for two weeks or more at about 25°C (e.g., about three weeks, about four weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, about 24 weeks, or more at about 25°C). In certain embodiments, the pharmaceutical composition is stable for about 4 weeks or more at about 25° C. In some embodiments, the pharmaceutical composition is stable for about 48 months or more at −20° C. (e.g., about 48 months, about 60 months, about 72 months, about 84 months, about 96 months, or more at −20° C.).

[0031] In some embodiments of any of the aspects and embodiments described above or enumerated herein, the pharmaceutical composition has a purity of about 85% or greater, e.g., as assessed by size-exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, the purity is about 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, e.g., 85% to 90%, 90% to 95%, or 95% to 100%, e.g., as assessed by SE-HPLC. In certain embodiments, the pharmaceutical composition has a purity of about 90% or greater, or about 95% or greater, as assessed by SE-HPLC. In some aspects, the pharmaceutical composition has a purity of about 95% or greater at about 36 months or greater as assessed by SE-HPLC at about 5° C. (e.g., 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater as assessed by SE-HPLC at about 5° C. or greater for about 36 months or greater, e.g., 85-90%, 90%-95%, or 95-100% as assessed by SE-HPLC at about 5° C.) In certain embodiments, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC at about 5° C. for about 42 months or greater, e.g., about 42 months, about 60 months, about 72 months, about 84 months, about 96 months, or longer at about 5° C.

[0032] In any of the preceding aspects and embodiments, the pharmaceutical composition has a saturation level of about 75% or greater as assessed by a non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay (e.g., about 76% or greater, about 77% or greater, about 78% or greater, about 79% or greater, about 80% or greater, about 81% or greater, about 82% or greater, about 83% or greater, about 84% or greater, about 85% or greater as assessed by a non-reducing CE-SDS assay). The pharmaceutical composition may have a purity of about 86% or greater, about 87% or greater, about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater, e.g., 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, as assessed by a non-reducing CE-SDS assay. In certain embodiments, the pharmaceutical composition has a purity of about 80% or greater as assessed by a non-reducing CE-SDS assay. For example, in some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay. In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay at about 5° C. over about 36 months (e.g., 85% or greater, about 86% or greater, about 87% or greater, about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater as assessed by a non-reducing CE-SDS assay, e.g., 85% to 90%, 90% to 95%, or 95% to 100% as assessed by a non-reducing CE-SDS assay at about 5° C. over about 36 months).In some embodiments, the pharmaceutical composition has a purity of about 85% or greater over about 42 months at about 5° C. as assessed by a non-reducing CE-SDS assay (e.g., about 85% or greater, about 86% or greater, about 87% or greater, about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater as assessed by a non-reducing CE-SDS assay, e.g., 85%-90%, 90%-95%, or 95%-100% as assessed by a non-reducing CE-SDS assay over about 42 months at about 5° C. In some embodiments, the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

[0033] In some embodiments, a pharmaceutical composition having any of the above shelf-life, purity, or stability characteristics is a DS. In other embodiments, a pharmaceutical composition having any of the above shelf-life, purity, or stability characteristics is a DP. In some embodiments, a pharmaceutical composition having any of the above-listed shelf-life or stability characteristics is frozen (e.g., stored at a temperature between -80°C and 2°C (e.g., about -40°C or 20°C) (e.g., about -40°C or -20°C)).

[0034] In some embodiments of any of the foregoing aspects and embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition does not contain a preservative. In some embodiments, the pharmaceutical composition is formulated for administration by injection after dilution with saline or a diluent (e.g., normal saline; e.g., normal saline; e.g., saline containing 0.45% or 0.9% (w / v) NaCl). In some embodiments, the liquid formulation is for dilution with an aqueous solution. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the saline contains sodium chloride (NaCl). In some embodiments, the saline solution contains 0.1 to 1.5% (e.g., 0.1 to 1.2%, 0.3 to 1.5%, 0.4 to 0.5%, 0.3 to 1%, 0.8 to 1%, 0.85 to 0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl.

[0035] In another aspect, the pharmaceutical composition of any of the foregoing aspects and embodiments is for use as a medicament.

[0036] In another aspect, the pharmaceutical composition of any of the foregoing aspects and embodiments is for use in treating or delaying the progression of cancer in a subject in need thereof (e.g., a human subject in need thereof).

[0037] In yet another aspect, the pharmaceutical composition of any of the preceding aspects and embodiments is for use in enhancing immune function in a subject with cancer.

[0038] In some embodiments, the cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, the NHL is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (DLBCL), germinal center B-cell-like (GCB) diffuse large B-cell lymphoma ( DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, and HHV8-associated multicentric Castleman disease Originates from large B-cell lymphoma, B-cell leukemia, follicular lymphoma (FL), in situ follicular neoplasia, mantle cell lymphoma (MCL), in situ mantle cell neoplasia, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic Selected from the group consisting of leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, childhood follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, and primary effusion lymphoma.In certain embodiments, the cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt's lymphoma (BL).

[0039] In some embodiments, mosunetuzumab is administered in a dose of about 0.1 mg to about 100 mg (e.g., 0.1 mg to 80 mg, 0.5 to 70 mg, 1 mg to 60 mg, 0.1 mg to 2 mg, 0.5 mg to 1.5 mg, 1 mg to 5 mg, 1.5 mg to 2.5 mg, 1 mg to 30 mg, 15 mg to 45 mg, 5 mg to 10 mg, 10 mg to 15 mg, 20 mg to 40 mg, 20 mg to 30 mg, 30 mg to 40 mg, 25 mg to 35 mg, 50 mg to 100 mg, 50 mg to 60 mg, 55 mg to 65 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg The mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg to about 60 mg, e.g., about 0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 13.5 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, or about 100 mg. In some embodiments, the method comprises administering mosunetuzumab to a subject at a dose of about 1 mg to about 60 mg. In certain embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, about 2 mg, about 6 mg, about 9 mg, about 13.5 mg, about 20 mg, about 30 mg, or about 60 mg. In some embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, 2 mg, 30 mg, or 60 mg.

[0040] In some embodiments, the pharmaceutical composition is diluted with saline before administration to a subject. In some embodiments, the saline is normal saline. In some embodiments, the saline contains sodium chloride (NaCl). In some embodiments, the saline contains 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl. In certain embodiments, the saline contains 0.45% or 0.9% (w / v) NaCl. In some embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml to about 0.3 mg / ml (e.g., about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.75 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, In certain embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0041] In some embodiments, the subject is to be co-administered with at least one additional therapeutic agent (e.g., 1, 2, 3, 4, or more additional therapeutic agents). In some embodiments, the at least one additional therapeutic agent includes a PD-1 axis-binding antagonist. In some embodiments, the PD-1 axis-binding antagonist is selected from the group consisting of a PD-L1-binding antagonist, a PD-1-binding antagonist, and a PD-L2-binding antagonist. In some embodiments, the PD-1 axis-binding antagonist is a PD-L1-binding antagonist (e.g., atezolizumab (MPDL3280A), MDX-1105 (BMS-936559; described in WO 2016 / 201425), and MEDI4736 (durvalumab)). In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (lambrolizumab), AMG404, REGN2810 (cemiplimab; LIBTAYO®), and AMP-224 (described in WO 2017 / 058780)). In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist (e.g., an antibody (e.g., an anti-PD-L2 antibody) or an immunoadhesin). In some embodiments, the at least one additional therapeutic agent comprises obinutuzumab, rituximab, an antibody-drug conjugate (ADC), a corticosteroid, or tocilizumab. In some embodiments, the at least one additional therapeutic agent comprises an ADC (e.g., an anti-CD79b ADC; e.g., polatuzumab vedotin). In some embodiments, the subject is a human.

[0042] In another aspect, the disclosure features a method of treating or delaying the progression of cancer in a subject in need thereof. In some embodiments, the method includes administering to the subject an effective amount of the pharmaceutical composition of any of the preceding aspects.

[0043] In another aspect, the disclosure features a method of enhancing immune function in a subject with cancer, e.g., by administering to the subject an effective amount of the pharmaceutical composition of any of the preceding aspects.

[0044] In some embodiments, the cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, the NHL is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (DLBCL), germinal center B-cell-like (GCB) diffuse large B-cell lymphoma ( DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, and HHV8-associated multicentric Castleman disease Originates from large B-cell lymphoma, B-cell leukemia, follicular lymphoma (FL), in situ follicular neoplasia, mantle cell lymphoma (MCL), in situ mantle cell neoplasia, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic Selected from the group consisting of leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, childhood follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, and primary effusion lymphoma.In certain embodiments, the cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt's lymphoma (BL).

[0045] In some embodiments, the NHL is DLBCL, GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL, or BL. In some embodiments, the NHL is FL or DLBCL. In some embodiments, the NHL is relapsed and / or refractory (R / R). In some embodiments, the NHL is R / R NHL. In some embodiments, the R / R FL has relapsed after or is refractory to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) prior systemic therapies. In some embodiments, the preceding one or more systemic therapies include an anti-CD20 monoclonal antibody. In some embodiments, the prior systemic therapy or multiple systemic therapies include an alkylating agent (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, altretamine, or thiotepa). In certain embodiments, the prior systemic therapy or multiple systemic therapies include both an anti-CD20 monoclonal antibody and an alkylating agent.

[0046] In some embodiments, mosunetuzumab is administered in a dose of about 0.1 mg to about 100 mg (e.g., 0.1 mg to 80 mg, 0.5 to 70 mg, 1 mg to 60 mg, 0.1 mg to 2 mg, 0.5 mg to 1.5 mg, 1 mg to 5 mg, 1.5 mg to 2.5 mg, 1 mg to 30 mg, 15 mg to 45 mg, 5 mg to 10 mg, 10 mg to 15 mg, 20 mg to 40 mg, 20 mg to 30 mg, 30 mg to 40 mg, 25 mg to 35 mg, 50 mg to 100 mg, 50 mg to 60 mg, 55 mg to 65 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg In some embodiments, the method comprises administering mosunetuzumab to a subject at a dose of about 1 mg to about 60 mg. In certain embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, about 2 mg, about 6 mg, about 9 mg, about 13.5 mg, about 20 mg, about 30 mg, or about 60 mg. In some embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, 2 mg, 30 mg, or 60 mg.

[0047] In some embodiments, the pharmaceutical composition is diluted with saline before administration to a subject. In some embodiments, the saline is normal saline. In some embodiments, the saline contains sodium chloride (NaCl). In some embodiments, the saline contains 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl. In certain embodiments, the saline contains 0.45% or 0.9% (w / v) NaCl. In some embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml to about 0.3 mg / ml (e.g., about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.75 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, In certain embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0048] In one embodiment, a pharmaceutical composition disclosed herein (e.g., comprising mosunetuzumab) is administered to a subject in a dosing regimen comprising at least three 21-day (±3 day) dosing cycles, wherein (a) the first 21-day dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of mosunetuzumab administered to the subject on or about days 1, 8 (±1 day), and 15 (±1 day) of the first dosing cycle, respectively, wherein C1D1 is administered in an amount of about 1 (b) the second administration cycle comprises a single dose (C2D1) of mosunetuzumab administered to the subject on or about day 1 of the second administration cycle, wherein C2D1 is about 60(±5) mg; (c) the third administration cycle comprises a single dose (C3D1) of mosunetuzumab administered to the subject on or about day 1 of the third administration cycle, wherein C3D1 is about 30(±3) mg. In some embodiments, the administration regimen comprises 1 to 14 additional administration cycles, each comprising an additional single dose of about 30(±0.5) mg of mosunetuzumab. In some embodiments, the administration regimen comprises 1 to 5 (e.g., 1, 2, 3, 4, or 5) additional administration cycles. In certain embodiments, the administration regimen comprises 5 additional administration cycles. In some embodiments, the subject is administered each additional single dose of mosunetuzumab on or about day 1 of each additional administration cycle.

[0049] In some embodiments, the subject is co-administered at least one additional therapeutic agent (e.g., 1, 2, 3, 4, or more additional therapeutic agents). In some embodiments, the at least one additional therapeutic agent includes a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist (e.g., atezolizumab (MPDL3280A), MDX-1105 (BMS-936559), and MEDI4736 (durvalumab)). In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (lambrolizumab), AMG404, REGN2810 (cemiplimab; LIBTAYO®), and AMP-224 (described in WO 2017 / 058780)). In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist (e.g., an antibody (e.g., an anti-PD-L2 antibody) or an immunoadhesin). In some embodiments, the at least one additional therapeutic agent comprises obinutuzumab, rituximab, an antibody-drug conjugate (ADC), a corticosteroid, or tocilizumab. In some embodiments, the at least one additional therapeutic agent comprises an ADC (e.g., an anti-CD79b ADC, e.g., polatuzumab vedotin).

[0050] In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the subject is a human.

[0051] Each and every embodiment may be combined unless the context clearly suggests otherwise. Any and all embodiments may be applied to any and all aspects of this disclosure unless the context clearly suggests otherwise.

[0052] Specific embodiments of the present disclosure will become apparent from the following more detailed description of certain preferred embodiments and the claims. [Brief explanation of the drawings]

[0053] [Figure 1] Figure 1 is a table summarizing considerations during delivery and use of various Phase III drug product (DP) formulations. [Figure 2] Figure 2 is a graph showing minimum PS20 concentration (% w / v) as a function of dose and DP protein concentration, as determined by an IV bag shake test in a 100 ml PO bag. The x-axis shows DP protein concentration (mg / ml) and the y-axis shows dose (mg). [Figure 3] Figure 3 is a graph showing the effect of various IV bag sizes and mosunetuzumab amounts on the minimum surfactant (PS20 or P188) concentration required to prevent aggregation and particle formation at 1 mg / ml DP. The first bar for each set of conditions on the x-axis (left to right) represents 5 mg or more of mosunetuzumab, the second bar represents 2 mg of mosunetuzumab, and the third bar represents 1 mg of mosunetuzumab. [Figure 4A] Figure 4A is a graph showing the kinetics of protein oxidation at methionine 257 (Met257) of mosunetuzumab (BTCT4465A) formulated with 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) of PS20 at 40°C. [Figure 4B] Figure 4B is a graph showing the kinetics of protein oxidation at methionine 257 of mosunetuzumab formulated with 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) of P188 at 40°C. [Figure 4C] FIG. 4C is a graph showing the kinetics of protein oxidation at methionine 257 of mosunetuzumab formulated with 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) of srPS20 at 40° C. [Figure 5]FIG. 5 is a graph showing the kinetics of protein oxidation at methionine 257 of mosunetuzumab formulated with 30 mM histidine (diamonds) or 10 mM histidine (squares) at 40° C. [Figure 6] 6 is a graph showing the percent oxidation of mosunetuzumab at methionine 257 in various formulations after up to 300,000 lux hours of ambient light exposure (light intensity of 5,500 lux). On the x-axis for each formulation, the first bar represents time=0, the second bar represents 24 hours, the third bar represents 54 hours, and the final (fourth) bar represents the dark control. [Figure 7A] Figure 7A is a graph showing the kinetics of hydrogen peroxide (HO) concentrations in various mosunetuzumab (BTCT4465A) compositions stored at 5°C for up to 12 months. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). HO concentrations were measured by the AMPLEX® Red assay. [Figure 7B] Figure 7B is a graph showing the kinetics of oxidation at methionine 257 in various mosunetuzumab compositions stored at 5°C for up to 12 months. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). Oxidation was measured by peptide mapping. [Figure 8A]Figure 8A is a graph showing the kinetics of hydrogen peroxide (HO) concentration in various mosunetuzumab compositions stored at 25°C for up to 6 months. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). HO concentration was measured by the AMPLEX® Red assay. [Figure 8B] Figure 8B is a graph showing the kinetics of oxidation at tryptophan 107 in the CD20 population for various mosunetuzumab compositions stored at 25°C for up to 6 months. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). Oxidation was measured by peptide mapping. [Figure 8C] Figure 8C is a graph showing the oxidation kinetics at methionine 257 for various mosunetuzumab compositions stored at 25°C for up to 6 months. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). Oxidation was measured by peptide mapping. [Figure 8D]8D is a graph showing the kinetics of high molecular weight species (HMWS) levels, as measured by SEC, in various mosunetuzumab compositions stored for up to 6 months at 25° C. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). [Figure 8E] 8E is a graph showing the kinetics of low molecular weight species levels, as measured by mCE-SDS, in various mosunetuzumab compositions stored for up to 6 months at 25° C. Mosunetuzumab compositions tested included composition alone (1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + HO (squares), composition + HO + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X), composition + HO + 5 mM methionine (light X), and composition + HO + 10 mM methionine (light X). [Figure 9A] Figure 9A is a graph showing the change in HMWS levels over time, as measured by SE-HPLC, in various mosunetuzumab formulations stored at 40°C and 75% relative humidity (RH) for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 9B] Figure 9B is a graph showing the change in monomer levels over time, as measured by SE-HPLC, in various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 9C] Figure 9C is a graph showing the change in LMWS levels over time, as measured by SE-HPLC, in various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 10A]Figure 10A is a graph showing the change in acidic variants over time, as measured by icIEF, in various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 10B] Figure 10B is a graph showing the change in the main peak over time as measured by icIEF for various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 10C] Figure 10C is a graph showing the change in basic variants over time, as measured by icIEF, in various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 11A] Figure 11A is a graph showing the change in sum of pre-peaks over time as measured by mCE-SDS for various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 11B] Figure 11B is a graph showing the change in the sum of the main peaks over time as measured by mCE-SDS for various mosunetuzumab formulations stored at 40°C and 75% RH for up to 1 month. Formulations F1-F5 are characterized in Table 6. [Figure 12] FIG. 12 is a graph showing the Donnan effect on pH values ​​of mosunetuzumab compositions. DETAILED DESCRIPTION OF THE INVENTION

[0054] I. Definition Unless otherwise specified, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0055] As used herein, the term "about" refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter preceded by "about" includes (and describes) embodiments that are directed to the value or parameter itself.

[0056] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an isolated peptide" means one or more isolated peptides.

[0057] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0058] The terms "pharmaceutical formulation" or "pharmaceutical composition" are used interchangeably herein and refer to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0059] A "pharmaceutically acceptable carrier" or "carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers or carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0060] The term "shelf life" refers to the length of time that a product (e.g., mosunetuzumab) can be stored without becoming unsuitable for use (e.g., by administration to a subject) or sale. In some embodiments, shelf life is the length of time that a composition (e.g., a pharmaceutical composition) is stable. For example, in some embodiments, the compositions herein have a shelf life of at least 36 months when stored at 5°C ± 3°C and protected from light.

[0061] A "stable" pharmaceutical formulation is one in which the protein (e.g., mosunetuzumab) therein essentially retains its physical stability and / or chemical stability and / or biological activity during storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity during storage (e.g., frozen storage). The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pub. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured over a selected period of time at a selected exposure dose and / or temperature. Stability may be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., by using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing ROS formation (e.g., by using a mild stress assay or a 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues in the protein (e.g., the Met residue in an antibody (such as mosunetuzumab)); assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact polypeptides; peptide map (e.g., trypsin or LYS-C) analysis; assessing the biological activity or target binding function of the protein (e.g., binding of an antibody to its antigen, e.g., binding of mosunetuzumab to T cells and / or target cells), etc.The instability may involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and / or Trp oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, and differential glycosylation, etc.

[0062] A protein (e.g., mosunetuzumab) "retains its physical stability" in a pharmaceutical formulation if it shows no or little signs of aggregation, precipitation, fragmentation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering or size exclusion chromatography.

[0063] A protein (e.g., mosunetuzumab) "retains its chemical stability" in a pharmaceutical formulation if the protein (e.g., mosunetuzumab) is still considered to retain its biological activity, as defined below, at a given time point. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein (e.g., mosunetuzumab). Chemical modifications can include protein oxidation, which can be assessed, for example, using tryptic peptide mapping, reverse-phase high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC / MS). Other types of chemical changes include charge changes of the protein (e.g., mosunetuzumab), which can be assessed, for example, by ion-exchange chromatography or icIEF.

[0064] A protein (e.g., mosunetuzumab) "retains its biological activity" in a pharmaceutical formulation if the biological activity of the protein (e.g., mosunetuzumab) at a given time is within about 20% (e.g., within about 10%) of the biological activity exhibited at the time the pharmaceutical formulation was prepared, as measured, for example, in a receptor binding assay.

[0065] As used herein, the "biological activity" of a protein (e.g., mosunetuzumab) refers to the ability of the protein to bind to its target, e.g., the ability of an antibody to bind to its antigen (e.g., the ability of mosunetuzumab to bind to T cells and / or target cells). This may further include biological responses that may be measured in vitro or in vivo. Such activity may be antagonistic or agonistic activity.

[0066] An "oxidation-susceptible" protein (e.g., mosunetuzumab) is a protein that contains one or more residues known to be susceptible to oxidation, such as, but not limited to, methionine (Met), cysteine ​​(Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr). For example, one or more methionine residues in mosunetuzumab (e.g., methionine 257, Met247, or M257) may be susceptible to oxidation.

[0067] The term "percent oxidation" refers to the percent of a protein (e.g., mosunetuzumab) in a formulation (e.g., pharmaceutical composition) that is oxidized at a particular amino acid residue, e.g., a Met residue. Percent oxidation can be determined, for example, by mass spectrometry (MS) of one or more tryptic peptides in which one or more particular oxidation-prone amino acid residues are present. Percent oxidation can be determined, for example, after an AAPH stress test, within 9 months, 12 months, 18 months, or 2 years of initial production of the protein (e.g., mosunetuzumab) or pharmaceutical composition thereof.

[0068] As used herein, the term "assessed by AAPH stress testing" refers to the determination of percent oxidation at a specific amino acid residue (e.g., a Met residue, e.g., Met257) by mass spectrometry analysis of tryptic peptides following formulation of a protein (e.g., mosunetuzumab) with AAPH (e.g., about 0 mM AAPH, about 1 mM AAPH, about 3 mM AAPH, about 3.5 mM AAPH, or about 5 mM AAPH) for about 24 hours at about 40°C in a formulation containing, for example, about 10 mg / ml mosunetuzumab, about 10 mM histidine acetate, about 240 mM sucrose, about 0.06 (w / v) polysorbate 20, and a pH of about 5.8. The stressed protein (e.g., mosunetuzumab) is digested with trypsin, and the digested peptides are subjected to LC-MS-MS to determine percent oxidation.

[0069] As used herein, "buffer" refers to a buffer solution (also referred to herein as a "buffering agent") that resists changes in pH due to the action of its acid-base complex components. In some embodiments, the buffer solution of the present disclosure has a pH in the range of about 4.5 to about 8. In some embodiments, the buffer solution has a pH in the range of about 5.5 to 6.1 (e.g., about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, or about 6.1), e.g., about pH 5.8. Exemplary buffering agents for use in the present disclosure include, but are not limited to, histidine (e.g., histidine acetate), acetate, phosphate, succinate, borate, citrate, tartrate, lactate, or combinations thereof. In some embodiments, the histidine is histidine acetate. In some embodiments, the phosphate is monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or a mixture thereof.

[0070] As used herein, "isotonicity agent" refers to an agent that can be added to a liquid (e.g., an aqueous solution) to adjust the isotonicity of the liquid. Osmolality refers to a measure of the osmotic pressure gradient between two solutions. In some embodiments, an isotonicity agent cannot pass through a semipermeable membrane (e.g., a semipermeable cell membrane) that would otherwise allow the liquid (e.g., an aqueous solution) or other components of the liquid (e.g., other solutes) to pass through. In some embodiments, an isotonicity agent is used to reduce local irritation by preventing osmotic shock at the application site. Exemplary isotonicity agents include carbohydrates (e.g., sucrose, glucose, dextrose, glycerol, glycerin, mannitol, and trehalose), amino acids, and salts (e.g., sodium chloride and potassium chloride).

[0071] As used herein, "surfactant" refers to a surface active agent, preferably a nonionic surfactant.Examples of surfactants herein include polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85); poloxamers (e.g., poloxamer 188); TRITON®; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl Examples of surfactants include betaine or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, New Jersey); polyethyl glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., PLURONIC®-type block copolymers, e.g., PLURONIC® F-68); and the like. In one embodiment, the surfactant herein is polysorbate 20 (PS20). In yet another embodiment, the surfactant herein is poloxamer 188 (P188).

[0072] A "preservative" is a compound that can be optionally included in a formulation to substantially reduce bacterial activity in the formulation, thereby, for example, facilitating the production of a multi-use formulation. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol; 3-pentanol, and m-cresol. In one embodiment, the preservative herein is benzyl alcohol. In some embodiments, the formulation does not contain a preservative.

[0073] The "molar ratio of surfactant to mosunetuzumab" (surfactant:antibody) is the ratio of surfactant to mosunetuzumab, with each component expressed as a molar concentration (also called molarity). Equation (1) shows this ratio: TIFF0007808151000001.tif18170

[0074] As used herein, "drug substance" or "DS" refers to a pharmaceutical composition formulated for storage, e.g., frozen storage, prior to administration to a subject. The DS may have a higher concentration of mosunetuzumab than the concentration of mosunetuzumab administered to a subject. Thus, in some instances, the DS is diluted prior to administration to a subject.

[0075] As used herein, "formulation" or "DP" refers to a pharmaceutical composition in its final configuration (e.g., in final vial configuration) ready to be administered to a subject. The concentration of mosunetuzumab in the DP may be the concentration administered to a subject. Alternatively, if the DP is intended to be administered with a diluent (e.g., saline; e.g., saline containing 0.45% or 0.9% (w / v) NaCl) or in combination with other therapeutic agents, the DP may be at a higher concentration than that administered to a subject.

[0076] As used herein, a "saline" solution refers to an aqueous solution that includes a salt (e.g., an ionic salt, e.g., sodium chloride (NaCl)). In some embodiments, the saline solution includes saline. In some embodiments, the saline solution includes 0.45% or 0.9% NaCl. In some embodiments, saline is used to dilute the liquid formulation prior to intravenous administration.

[0077] Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references, such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), PCR Protocols: A Guide to Methods and Applications (Innis, et al., 1990, Academic Press, San Diego, CA), and Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0078] Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and / or parameters unless otherwise specified. Therefore, before the present methods and uses are described, it should be understood that the present disclosure is not limited to specific methodologies, protocols, cell lines, animal species or genera, constructs, and reagents, which may, of course, vary. It should also be understood that the terminology used herein is intended to describe particular embodiments only and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0079] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., mosunetuzumab), and antibody fragments, so long as they exhibit the desired antigen-binding activity (e.g., antigen-binding fragments of antibodies).

[0080] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0081] "Binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. A binding domain can be part of a molecule such as an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, or a chimeric antibody), an antibody fragment, or portion thereof (e.g., a Fab fragment, a Fab'2, an scFv antibody, a SMIP, a domain antibody, a diabody, a minibody, an scFv-Fc, an affibody, a nanobody, or the VH and / or VL domains of an antibody), a receptor, a ligand, an aptamer, or other molecule with an identified binding partner.

[0082] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., supra); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b) and / or (c) comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0083] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th See, W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of an antibody that binds to that antigen and then screened against a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0084] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., supra.

[0085] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0086] "Percent (%) amino acid sequence identity" or "percent (%) sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0087] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it can be written as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with, or against, amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically indicated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0088] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0089] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., supra. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III in Kabat et al., supra.

[0090] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0091] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0092] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or of an antibody derived from a non-human source utilizing the human antibody repertoire, or to the sequence encoding another human antibody. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, such as phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized XENOMOUSE™, which have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies produced by human B cell hybridoma technology.

[0093] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (which may contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation, and such variants are typically present in minor amounts). Each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen, in contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes). Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0094] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0095] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 146,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or light chain variable domain, followed by one constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0096] As used herein, the term "half antibody" refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain.

[0097] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of testing methods for antibody purification, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0098] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] As used herein, the term "cluster of differentiation 3" or "CD3," unless otherwise specified, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynomolgus monkeys)) and rodents (e.g., mice and rats), including, for example, the CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. Examples of CD3 include the 207 amino acid long human CD3ε protein (NCBI RefSeq No. NP_000724), the 182 amino acid long human CD3γ protein (NCBI RefSeq No. NP_000064), the 198 amino acid long cynomolgus monkey CD3ε protein (NCBI RefSeq No. NP_1270544.1), and the 181 amino acid long cynomolgus monkey CD3γ protein (NCBI RefSeq No. NP_1270839.1).

[0100] As used herein, the term "cluster of differentiation 20" or "CD20," unless otherwise indicated, refers to any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD20, as well as any form of CD20 that results from processing within the cell. The term also encompasses naturally occurring variants of CD20, including, for example, splice variants or allelic variants. CD20 includes, for example, the human CD20 protein (see, e.g., NCBI Reference SEQ ID NOs: NP_068769.2 and NP_690605.1), which is, for example, 297 amino acids in length and can be produced from, for example, a mutant mRNA transcript lacking a portion of the 5' UTR (see, e.g., NCBI Reference SEQ ID NO: NM_021950.3), or a longer mutant mRNA transcript (see, e.g., NCBI Reference SEQ ID NO: NM_152866.2).

[0101] The terms "anti-CD20 / anti-CD3 antibody," "anti-CD20 / anti-CD3 bispecific antibody," "anti-CD20 / anti-CD3 TDB," or variants thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that can bind to CD20 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20 and / or CD3. In one embodiment, the extent of binding of an anti-CD20 / anti-CD3 antibody to unrelated non-CD3 and / or non-CD20 proteins is less than about 10% of the binding of the antibody to CD3 and / or CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D) binds to CD20 and CD3. In some embodiments, the anti-CD20 / anti-CD3 antibody is mosunetuzumab. Mosunetuzumab (also known as BTCT4465A or RG7828) is defined by the International Nonproprietary Names for Pharmaceutical Substances (INN) List 117 (WHO Drug Information, Vol. 31, No. 2, 2017, pp. 304-305). In some examples, mosunetuzumab is an anti-CD20 / anti-CD3 bispecific antibody (anti-CD20 / anti-CD3 T cell-dependent bispecific antibody) described in PCT Application Publication No. 2015 / 09539, the entire contents of which are incorporated herein by reference.

[0102] A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0103] As used herein, "administering" refers to a method of providing a dosage of mosunetuzumab or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising mosunetuzumab) to a subject. Pharmaceutical compositions utilized in the methods described herein can be administered, for example, intravenously, intradermally, intramuscularly, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraumbilically, intraocularly, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized bath target cells, directly, by catheter, by perfusion, in a cream, or in a lipid composition. The method of administration can vary depending on various factors, such as the pharmaceutical composition being administered and the severity of the condition, disease, or disorder (e.g., cancer) being treated.

[0104] As used herein, a "week" is 7 days ± 2 days. As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, remission or alleviation of symptoms, and recovery or improved prognosis. In some embodiments, the pharmaceutical compositions of the present disclosure are used to delay the onset of disease or slow the progression of disease.

[0105] As used herein, "enhancing immune function" in a subject means inducing, eliciting, stimulating, maintaining, or amplifying an innate or adaptive immune response. In some embodiments, enhancing immune function includes enhancing T cell function. In some embodiments, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Methods for measuring this enhancement are well known to those of skill in the art.

[0106] As used herein, "enhancing T cell function" refers to inducing, triggering, or stimulating T cells to maintain or amplify their biological function, or to renew or reactivate exhausted or inactive T cells. Examples of enhanced T cell function include increased secretion of gamma interferon from CD8+ T cells, increased proliferation, or enhanced antigen responsiveness (e.g., viral, pathogen, or tumor clearance) compared to pre-intervention levels. In some embodiments, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, or 500%. The manner of measuring this enhancement is known to those skilled in the art.

[0107] As used herein, "delaying the progression" of cancer means postponing, preventing, delaying, retarding, stabilizing, and / or postponing the onset of cancer. This delay can be of various lengths of time, depending on the cancer being treated and / or the individual's medical history. As will be apparent to those skilled in the art, a sufficient or significant delay can actually encompass prevention, in that the individual does not develop cancer. For example, late-stage cancer, such as the onset of metastasis, can be delayed.

[0108] "Reducing" or "inhibiting" refers to the ability to cause an overall decrease, for example, of 20% or more, 50% or more, or 75%, 85%, 90%, 95%, or more. In certain embodiments, reducing or inhibiting may refer to antibody effector functions mediated by the antibody Fc region, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0109] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including conditions that predispose a mammal to the disorder in question.

[0110] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaving cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as lipomatosis, edema (such as associated with brain tumors), Meige syndrome, brain, and head and neck cancer and associated metastases.In other embodiments, the cancer is excluding Hodgkin's lymphoma, but is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia Selected from mature B-cell cancers including hematologic variants, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, chronic inflammation-associated DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, and primary effusion lymphoma. Selected from mature B-cell cancers including unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0111] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0112] As used herein, the term "tumor antigen" can be understood as an antigen presented on tumor cells. These antigens may be presented on the cell surface with the extracellular portion often associated with the transmembrane and cytoplasmic portions of the molecule. These antigens are presented only by tumor cells and never by normal cells. Tumor antigens may be expressed only on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. Tumor antigens presented by tumor cells and normal cells are more common and are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be accessible to antibodies that bind to tumor cells because the structure of tumor tissue is not small compared to normal tissue.

[0113] "Effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0114] An "effective amount" of a pharmaceutical composition, e.g., a pharmaceutical composition comprising mosunetuzumab, is at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, such as a measurable improvement in cancer. The effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. An effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. For prophylactic use, beneficial or desired results include results such as a reduced risk, reduced severity, or delayed onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifesting during disease development. For therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms attributable to the disease, an improvement in the quality of life of a person suffering from the disease, a reduction in the dose of other drugs required to treat the disease, an enhancement of the effect of another drug (e.g., by targeting), a delay in disease progression, and / or an increase in survival time. In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing or desirably stopping) the infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing or desirably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, directly or indirectly. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount when a desired result is obtained or achieved in combination with one or more other agents.

[0115] The term "package insert" is used to refer to instructions customarily included in commercially available packaging of a therapeutic product that contain information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0116] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis-binding partner with any one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0117] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-1 with one or more binding partners, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate PD-1-mediated signaling, reducing the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab), as described herein. In another specific aspect, the PD-1 binding antagonist is MK-3475 (lambrolizumab), as described herein. In another specific aspect, the PD-1 binding antagonist is AMG404, as described herein. In another specific aspect, the PD-1 binding antagonist is REGN2810 (cemiplimab; LIBTAYO®), as described herein. In another specific embodiment, the PD-1 binding antagonist is AMP-224, as described herein and in WO 2017 / 058780.

[0118] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, e.g., PD-1, B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In particular aspects, PD-L1 binding antagonists inhibit the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed in response to T lymphocyte-mediated signaling via PD-L1, thereby alleviating dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is atezolizumab (MPDL3280A), as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MDX-1105 (BMS-936559), as described herein and in WO 2016 / 201425. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 (durvalumab), as described herein.

[0119] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling that occurs as a result of the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a particular aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling that results from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed in response to T lymphocyte-mediated signaling through PD-L2, thereby alleviating the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0120] II. Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising mosunetuzumab and uses thereof, for example, for the treatment of cancer (e.g., hematological cancer). The pharmaceutical compositions of the present disclosure may be formulated to support relatively low concentrations of mosunetuzumab.

[0121] In one aspect, the present disclosure provides a pharmaceutical composition comprising mosunetuzumab, a surfactant (e.g., polysorbate 20 (PS20)), methionine, a buffer, and a carrier, wherein the PS20 concentration is 0.01% to 0.1% weight / volume (w / v), the methionine concentration is 1 mM to 50 mM, and the buffer concentration is 5 mM to 20 mM.

[0122] The pharmaceutical composition may contain mosunetuzumab at a concentration of about 0.5 mg / ml to about 2 mg / ml, e.g., about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, or about 2.0 mg / ml. In certain embodiments, the concentration of mosunetuzumab is about 1 mg / ml.

[0123] In some examples, the molar ratio of surfactant (e.g., PS20) to mosunetuzumab is 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 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 or less, or 1 or less, e.g., 0.5 to 100, 0.5 to 50, 0.5 to 10, 0.5 to 5, 0.5 to 1, 1 to 5, 2 to 4, 5 to 100, 10 to 70, 10 to 50, 5 to 25, 10 to 30, or 50 to 100, 60 to 80, or 65 to 75. In certain embodiments, the molar ratio of surfactant to mosunetuzumab is 50 to 100. In some embodiments, the molar ratio of surfactant to mosunetuzumab is 50-100, 60-80, or 65-75, e.g., about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, the DP may have a molar ratio of surfactant (e.g., PS20) to mosunetuzumab of 50-100. For example, in certain embodiments, the DP may have a molar ratio of surfactant (e.g., PS20) to mosunetuzumab of about 71. In some embodiments, the DP may have a molar ratio of surfactant (e.g., P188) to mosunetuzumab of 5-25. For example, in certain embodiments, the DP may have a molar ratio of surfactant (e.g., PS20) to mosunetuzumab of about 17.

[0124] The disclosed pharmaceutical composition comprises a surfactant.Any suitable surfactant can be used.In some embodiments, the surfactant is preferably a non-ionic surfactant (e.g., polysorbate (polyoxyethylene(n) sorbitan monolaurate), poloxamer, polyoxyethylene alkyl ether, alkylphenyl polyoxyethylene ether, or a combination thereof). In some embodiments, the nonionic surfactant is a polysorbate (e.g., polysorbate 20 (PS20; e.g., polyoxyethylene (20) sorbitan monolaurate, e.g., TWEEN 20®; e.g., SuperRefined™ PS20 (PS20 that has been subjected to a proprietary flash chromatography process for higher purity and is available from Avantor Performance Materials, LLC, Center Valley, Pennsylvania, USA)) or polysorbate 80 (PS80; e.g., polyoxyethylene (20) sorbitan monooleate, e.g., TWEEN 80®; e.g., SuperRefined™ PS80 (Avantor)). In certain embodiments, the polysorbate is polysorbate 20 (PS20). In other embodiments, the nonionic surfactant is a poloxamer (e.g., poloxamer 188, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)).

[0125] Any suitable concentration of surfactant may be used. The concentration of surfactant in the pharmaceutical composition may be selected based on the desired molar ratio of surfactant to mosunetuzumab. In some embodiments of any of the pharmaceutical compositions described herein, the concentration of surfactant (e.g., PS20 or P188) is about 0.001% (w / v) to about 2% (w / v), e.g., about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, or about 0.08% (w / v). %, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is about 0.05% (w / v) to about 0.1% (w / v). In certain embodiments, the surfactant is PS20 and the concentration of PS20 is about 0.06% (w / v). In certain embodiments, the surfactant is P188 and the concentration of P188 is about 0.1% (w / v).

[0126] Any pharmaceutical composition described herein may include a stabilizer. Any suitable stabilizer may be used. For example, in some embodiments, the stabilizer is thiosorbitol, ascorbic acid, monothioglycerol, cyclodextrin, Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), pyridoxine, mannitol, a metal chelator, an amino acid, or a combination thereof. In some embodiments, the stabilizer is an amino acid. In some embodiments, the amino acid is methionine, cysteine, tryptophan, or a combination thereof. In certain embodiments, the amino acid is methionine.

[0127] Any suitable concentration of stabilizer (e.g., methionine) may be used. For example, in some embodiments of any of the aforementioned pharmaceutical compositions, the concentration of stabilizer (e.g., methionine) may be about 0.01 mM to about 50 mM, for example, about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, 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, 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, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM. In some embodiments, the concentration of the stabilizer (e.g., methionine) is about 1 mM to about 50 mM, about 2 mM to about 50 mM, about 3 mM to about 50 mM, about 4 mM to about 50 mM, about 5 mM to about 50 mM, about 6 mM to about 50 mM, about 7 mM to about 50 mM, about 8 mM to about 50 mM, about 9 mM to about 50 mM, about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 1 mM to about 40 mM, about 2 mM to about 40 mM, about 3 mM to about 40 mM, about 4 mM to about 40 mM, about 5 mM to about 40 mM, about 5 mM to about 40 mM, about 6 mM to about 50 mM, or about 7 mM to about 50 mM. M ~ about 40mM, about 7mM - about 40mM, about 8mM - about 40mM, about 9mM - about 40mM, about 10mM - about 40mM, about 15mM - about 40mM, Approximately 20mM to approximately 40mM, approximately 25mM to approximately 40mM, approximately 30mM to approximately 40mM, approximately 1mM to approximately 30mM, approximately 2mM to approximately 30mM, approximately 3mM to approximately 3 0mM, about 4mM to about 30mM, about 5mM to about 30mM, about 6mM to about 30mM, about 7mM to about 30mM, about 8mM to about 30mM, about 9mM to about 30mM, about 10mM to about 30mM, about 11mM to about 30mM, about 12mM to about 30mM, about 13mM to about 30mM, about 14mM to about 30mM,Approximately 15mM to approximately 30mM, approximately 20mM to approximately 30mM, approximately 25mM to approximately 30mM, approximately 1mM to approximately 20mM, approximately 2mM to approximately 20mM, approximately 3mM to Approximately 20mM, approximately 4mM to approximately 20mM, approximately 5mM to approximately 20mM, approximately 6mM to approximately 20mM, approximately 7mM to approximately 20mM, approximately 8mM to approximately 20mM, approximately 9m M ~ about 20mM, about 10mM - about 20mM, about 11mM - about 20mM, about 12mM - about 20mM, about 13mM - about 20mM, about 14mM - about 2 0mM, about 15mM to about 20mM, about 1mM to about 15mM, about 2mM to about 15mM, about 3mM to about 15mM, about 4mM to about 15mM, about 5mM to about 15 mM, about 6 mM to about 15 mM, about 7 mM to about 15 mM, about 8 mM to about 15 mM, about 9 mM to about 15 mM, about 10 mM to about 15 mM, about 11 mM to about 15 mM, about 12 mM to about 15 mM, about 13 mM to about 15 mM, about 14 mM to about 15 mM, about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, about 9 mM to about 10 mM, about 1 mM to about 5 mM, about 2 mM to about 5 mM, about 3 mM to about 5 mM, or about 4 mM to about 5 mM.

[0128] In some embodiments, the concentration of methionine is between 2.5 mM and 20 mM (e.g., between 2.5 mM and 5 mM, between 5 mM and 7.5 mM, between 7.5 mM and 10 mM, between 10 mM and 12.5 mM, between 12.5 mM and 15 mM, between 15 mM and 17.5 mM, or between 17.5 mM and 20 mM, e.g., between 3 mM and 18 mM, between 4 mM and 18 mM, or between 17.5 mM and 20 mM). In certain embodiments, the methionine concentration is about 10 mM.

[0129] In a pharmaceutical composition containing mosunetuzumab, oxidation of methionine at position 257 of the Fc region is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%; e.g., 0-9%, 0-8%, 0-7%, 0-6%, 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 1-5%, 1-10%, 2-9%, 3-8%, or 4-7%; e.g., about 10%, about 9%, about 8%, about 7%, about 6%, about 4%, about 4%, about 3%, about 2%, about 1%, or about 0%) over a period of 2 weeks at 40°C. In a specific embodiment, oxidation of methionine at position 257 of the Fc region is about 6% or less (e.g., 0-6%) over a period of 2 weeks at 40°C.

[0130] Any of the aforementioned compositions (e.g., pharmaceutical compositions) may further comprise a buffering agent. Any suitable buffering agent may be used. In some embodiments, the buffering agent is histidine, acetate, phosphate, succinate, or a combination thereof. In some embodiments, the histidine is histidine acetate. Alternative buffering agents include histidine hydrochloride (histidine HCl), histidine acetate, monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or a mixture thereof.

[0131] In a specific example, the concentration of the buffer (e.g., histidine, e.g., histidine acetate) is 5 mM to 20 mM. For example, the buffer can be 5 mM to 10 mM, 10 mM to 15 mM, or 15 mM to 20 mM, e.g., 6 mM to 18 mM, 7 mM to 16 mM, 8 mM to 15 mM, 9 mM to 12 mM, or 8 mM to 12 mM, e.g., 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 certain examples, the concentration of the buffering agent (e.g., histidine, e.g., histidine acetate) can be, for example, 8 mM to 12 mM, e.g., about 8 mM, about 9 mM, about 10 mM, about 11 mM, or about 12 mM. In certain embodiments, the concentration of the buffering agent (e.g., histidine, e.g., histidine acetate) is about 10 mM. In certain embodiments, the buffering agent is L-histidine acetate and is present at a concentration of about 10 mM.

[0132] In some embodiments, the pharmaceutical composition comprises a tonicity agent, such as a carbohydrate (e.g., sucrose, glucose, dextrose, glycerol, glycerin, mannitol, and trehalose), an amino acid, or a salt (e.g., sodium chloride and potassium chloride). In embodiments in which the tonicity agent is a sugar, the sugar can be, for example, sucrose, glucose, glycerol, or trehalose. In certain embodiments, the sugar is sucrose. The concentration of the tonicity agent (e.g., a sugar, such as sucrose) can be from about 100 mM to about 500 mM. For example, the concentration of the isotonic agent (e.g., a sugar, such as sucrose) may be 100 mM to 120 mM, 120 mM to 140 mM, 140 mM to 160 mM, 160 mM to 180 mM, 180 mM to 200 mM, 200 mM to 220 mM, 220 mM to 240 mM, 240 mM to 260 mM, 260 mM to 280 mM, 280 mM to 300 mM, 300 mM to 320 mM, 320 mM to 340 mM, 340 mM to 360 mM, 360 mM to 380 mM, 380 mM to 400 mM, 400 mM to 420 mM, 4 The concentration of the tonicity agent is 20 mM to 440 mM, 440 mM to 460 mM, 460 mM to 480 mM, or 480 mM to 500 mM, e.g., 100 mM to 400 mM, 150 mM to 350 mM, or 200 mM to 300 mM, e.g., about 100 mM, about 150 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM. In some embodiments, the concentration of the tonicity agent is about 240 mM. In a particular embodiment, the tonicity agent is sucrose and is present at a concentration of about 240 mM.

[0133] The pH of the pharmaceutical composition may be any suitable pH. In some embodiments, the pharmaceutical composition has a pH of about 4.5 to about 8 (e.g., 4.5 to 5.0, 5.0 to 5.5, 5.5 to 6.0, 6.0 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0), e.g., about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, etc. The pharmaceutical composition has a pH in the range of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the pharmaceutical composition has a pH of 5.5 to 6.1. In certain embodiments, the pharmaceutical composition has a pH of about 5.8.

[0134] In addition to the specific ingredients described above, the pharmaceutical compositions of the present disclosure may also be prepared by mixing mosunetuzumab having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A., ed. (1980)) in the form of a lyophilized preparation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; carbohydrates such as monosaccharides (e.g., glucose, mannose), disaccharides (e.g., sucrose, trehalose), or polysaccharides (e.g., dextrin), or sugar alcohols such as mannitol or sorbitol; chelating agents such as ethylenediaminetetraacetic acid (EDTA); salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), for example, human soluble PH-20 hyaluronidase glycoproteins, for example, rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0135] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation containing a histidine acetate buffer.

[0136] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., a liquid formulation for infusion, a liquid formulation for injection, a liquid formulation for dilution, etc.). In certain embodiments, the pharmaceutical composition is a liquid formulation for dilution. In certain embodiments, the liquid formulation for dilution is supplied in a container having a volume of about 50 ml (e.g., about 40 ml, about 45 ml, about 46 ml, about 47 ml, about 48 ml, about 49 ml, about 50 ml, about 51 ml, about 52 ml, about 53 ml, about 54 ml, about 55 ml, or about 60 ml). In some embodiments, the volume of the liquid formulation for dilution is 20 to 40 ml (e.g., 20 to 30 ml, 30 to 40 ml, 20 to 35 ml, 25 to 40 ml, 25 to 35 ml, or 28 to 32 ml; e.g., about 20 ml, about 25 ml, about 26 ml, about 27 ml, about 28 ml, about 29 ml, about 30 ml, about 31 ml, about 32 ml, about 33 ml, about 34 ml, about 35 ml, or about 40 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 30 ml. In another specific embodiment, the liquid formulation for dilution is supplied in a container having a volume of about 2 ml (e.g., about 1 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, about 2 ml, about 2.1 ml, about 2.2 ml, about 2.3 ml, about 2.4 ml, about 2.5 ml, or about 3 ml). In some embodiments, the volume of the liquid formulation for dilution is 0.2 to 2 ml (e.g., 0.2 to 1.5 ml, 0.5 to 2 ml, 0.5 to 1 ml, or 0.8 to 1.2 ml; e.g., about 0.2 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 1.1 ml, about 1.2 ml, about 1.3 ml, about 1.4 ml, about 1.5 ml, or about 2 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 1 ml.

[0137] In some embodiments, the liquid formulation is for dilution with a diluent. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the liquid formulation is for dilution with saline. In some embodiments, the saline comprises sodium chloride (NaCl). In some embodiments, the saline comprises 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl.

[0138] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and / or an antihormonal agent, such as those mentioned hereinabove). Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0139] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions, for example, by microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).

[0140] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0141] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0142] Any pharmaceutical composition described herein may have a shelf life of at least about 12 months (e.g., at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months) when stored at 5°C ± 3°C and protected from light. In some embodiments, the pharmaceutical composition has a shelf life of at least 36 months when stored at 5°C ± 3°C and protected from light. In some embodiments, the composition has a shelf life of at least 42 months when stored at 5°C ± 3°C and protected from light. In some embodiments, the composition has a shelf life of at least 48 months when stored at 5°C ± 3°C and protected from light.

[0143] In some embodiments, the shelf life when stored at 5°C ± 3°C and protected from light is from about 1 month to about 72 months (e.g., about 1 month, about 5 months, about 10 months, about 15 months, about 20 months, about 24 months, about 25 months, about 30 months, about 35 months, about 40 months, about 45 months, about 48 months, about 50 months, about 55 months, about 60 months, about 65 months, about 70 months, or about 72 months). In some embodiments, the shelf life when stored at 5°C ± 3°C and protected from light is about 1 month to about 72 months, about 1 month to about 70 months, about 1 month to about 65 months, about 1 month to about 60 months, about 1 month to about 55 months, about 1 month to about 50 months, about 1 month to about 48 months, about 1 month to about 45 months, about 1 month to about 40 months, about 1 month to about 35 months, about 1 month to about 30 months, about 1 month to about 25 months, about 1 month to about 24 months, about 1 month to about 20 months, about 1 month to about 18 months, about 1 month to about 20 ... Approximately 15 months, approximately 1 month to approximately 12 months, approximately 1 month to approximately 9 months, approximately 1 month to approximately 6 months, approximately 1 month to approximately 3 months, approximately 5 months to approximately 72 months, approximately 5 months to approximately 70 months, approximately 5 months to approximately 65 months, approximately 5 months to approximately 60 months, approximately 5 months to approximately 55 months, approximately 5 months to approximately 50 months, approximately 5 months to approximately 48 months, approximately 5 months to approximately 45 months, approximately 5 months to approximately 40 months, approximately 5 months to approximately 35 months, approximately 5 months to approximately 30 months, approximately 5 months to approximately 25 months, approximately 5 months to approximately 24 months, approximately 5 months to approximately 20 months, approximately 5 months to approximately 18 months months, about 5 months to about 15 months, about 5 months to about 12 months, about 5 months to about 9 months, about 5 months to about 6 months, about 10 months to about 72 months, about 10 months to about 70 months, about 10 months to about 65 months, about 10 months to about 60 months, about 10 months to about 55 months, about 10 months to about 50 months, about 10 months to about 48 months, about 10 months to about 45 months, about 10 months to about 40 months, about 10 months to about 35 months, about 10 months to about 30 months, about 10 months to about 25 months, about 10 months to about 24 months, about 10 months to about 20 months, approximately 10 months to approximately 18 months, approximately 10 months to approximately 15 months, approximately 10 months to approximately 12 months, approximately 12 months to approximately 72 months, approximately 12 months to approximately 70 months, approximately 12 months to approximately 65 months, approximately 12 months to approximately 60 months, approximately 12 months to approximately 55 months, approximately 12 months to approximately 50 months, approximately 12 months to approximately 48 months, approximately 12 months to approximately 45 months, approximately 12 months to approximately 40 months, approximately 12 months to approximately 35 months, approximately 12 months to approximately 30 months, approximately 12 months to approximately 25 months, approximately 12 months to approximately 24 months, approximately 12 months to approximately 20 months,Approximately 12 months to 18 months, approximately 12 months to 15 months, approximately 18 months to 72 months, approximately 18 months to 70 months, approximately 18 months to 65 months, approximately 18 months to 60 months, approximately 18 months to 55 months, approximately 18 months to 50 months, approximately 18 months to 48 months, approximately 18 months to 45 months, approximately 18 months to 40 months, approximately 18 months to 35 months, approximately 18 months to 30 months, approximately 18 months to 25 months, approximately 18 months to 24 months, approximately 18 months to 20 months, approximately 24 months to 72 months, approximately 24 months to 70 months, approximately 24 months to 65 months, approximately 24 months to 60 months, approximately 24 months to 55 months, about 24 months to about 50 months, about 24 months to about 48 months, about 24 months to about 45 months, about 24 months to about 40 months, about 24 months to about 35 months, about 24 months to about 30 months, about 24 months to about 25 months, about 30 months to about 72 months, about 30 months to about 70 months, about 30 months to about 65 months, about 30 months to about 60 months, about 30 months to about 55 months, about 30 months to about 50 months, about 30 months to about 48 months, about 30 months to about 45 months, about 30 months to about 40 months, about 30 months to about 35 months, about 30 months to about 36 months, about 36 months to about 72 months, about 36 months to about 70 months, about 36 months to Approximately 65 months, approximately 36 months to approximately 60 months, approximately 36 months to approximately 55 months, approximately 36 months to approximately 50 months, approximately 36 months to approximately 48 months, approximately 36 months to approximately 45 months, approximately 36 months to approximately 40 months, approximately 40 months to approximately 72 months, approximately 40 months to approximately 70 months, approximately 40 months to approximately 65 months, approximately 40 months to approximately 60 months, approximately 40 months to approximately 55 months, approximately 40 months to approximately 50 months, approximately 40 months to approximately 48 months, approximately 40 months to approximately 45 months, approximately 42 months to approximately 72 months, approximately 42 months to approximately 70 months, approximately 42 months to approximately 65 months, approximately 42 months to approximately 60 months, approximately 42 months to approximately 55 months, approximately 42 months to approximately 50 months, approximately 42 months to approximately 42 months months to about 48 months, about 42 months to about 45 months, about 46 months to about 72 months, about 46 months to about 70 months, about 46 months to about 65 months, about 46 months to about 60 months, about 46 months to about 55 months, about 46 months to about 50 months, about 46 months to about 48 months, about 48 months to about 72 months, about 48 months to about 70 months, about 48 months to about 65 months, about 48 months to about 60 months, about 48 months to about 55 months, about 48 months to about 50 months, about 50 months to about 72 months, about 50 months to about 70 months, about 50 months to about 65 months, about 50 months to about 60 months, about 50 months to about 55 months, about 55 months to about 72 months,Approximately 55 months to approximately 70 months, approximately 55 months to approximately 65 months, approximately 55 months to approximately 60 months, approximately 60 months to approximately 72 months, approximately 60 months to approximately 70 months, or approximately 60 months to approximately 65 months.

[0144] A stable pharmaceutical composition may contain, for example, 1,000 or fewer particles having a diameter of 2 μm or greater per ml. For example, the pharmaceutical composition may contain 900 or fewer, 800 or fewer, 700 or fewer, 600 or fewer, 500 or fewer, 400 or fewer, 300 or fewer, 200 or fewer, or 100 or fewer particles having a diameter of 2 μm or greater per ml (e.g., 0 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1,000 particles having a diameter of 2 μm or greater per ml). In some embodiments, the carrier is water.

[0145] Additionally or alternatively, the stable pharmaceutical composition may have a purity of about 85% or greater. In some embodiments, the purity is about 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, e.g., 85% to 90%, 90% to 95%, or 95% to 100%, as assessed, for example, by size-exclusion high performance liquid chromatography (SE-HPLC). In certain embodiments, the pharmaceutical composition has a purity of about 90% or greater, as assessed by SE-HPLC, or about 95% or greater, as assessed by SE-HPLC. In some embodiments, the pharmaceutical composition has a purity of about 95% or greater at about 36 months or more as assessed by SE-HPLC at about 5° C. (e.g., 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater as assessed by SE-HPLC at about 5° C. or more for about 36 months or more, e.g., 85-90%, 90%-95%, or 95-100% as assessed by SE-HPLC at about 5° C.) In particular examples, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC at about 5° C. for about 42 months or more, e.g., about 42 months, about 60 months, about 72 months, about 84 months, about 96 months, or longer at about 5° C.

[0146] In some examples, the present disclosure provides a method for producing a soluble soluble cellulose membrane comprising a soluble cellulose membrane comprising at least about 75% soluble cellulose membrane protein as assessed by a non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay (e.g., at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% soluble cellulose membrane protein as assessed by a non-reducing CE-SDS assay). The present invention provides pharmaceutical compositions having a purity of about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, or about 99% or greater, e.g., 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, as assessed by a non-reducing CE-SDS assay. In certain embodiments, the pharmaceutical compositions have a purity of about 80% or greater as assessed by a non-reducing CE-SDS assay. For example, in some embodiments, the pharmaceutical compositions have a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay. In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay at about 5° C. over about 36 months (e.g., 85% or greater, about 86% or greater, about 87% or greater, about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater as assessed by a non-reducing CE-SDS assay, e.g., 85% to 90%, 90% to 95%, or 95% to 100% as assessed by a non-reducing CE-SDS assay at about 5° C. over about 36 months).In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay at about 5° C. over about 42 months (e.g., 85% or greater, about 86% or greater, about 87% or greater, about 88% or greater, about 89% or greater, about 90% or greater, about 91% or greater, about 92% or greater, about 93% or greater, about 94% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater as assessed by a non-reducing CE-SDS assay, e.g., 85% to 90%, 90% to 95%, or 95% to 100% as assessed by a non-reducing CE-SDS assay over about 42 months at about 5° C.).

[0147] III. Mosunetuzumab Mosunetuzumab may incorporate any of the features, alone or in combination, as described herein.

[0148] In some examples, mosunetuzumab is an anti-CD20 antibody having a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from: (1) an HVR-H1 comprising the amino acid sequence of GYTFTSYNMH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the amino acid sequence of VVYYSNSYWYFDV (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acid sequence of RASSSVSYMH (SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acid sequence of APSNLAS (SEQ ID NO: 5); or (f) an HVR-L3 comprising the amino acid sequence of QQWSFNPPT (SEQ ID NO: 6). and (2) an anti-CD3 arm having a second binding domain comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of NYYIH (SEQ ID NO: 9); (b) HVR-H2 comprising the amino acid sequence of WIYPGDGNTKYNEKFKG (SEQ ID NO: 10); (c) HVR-H3 comprising the amino acid sequence of DSYSNYYFDY (SEQ ID NO: 11); (d) HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO: 12); (e) HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 13); and (f) HVR-L3 comprising the amino acid sequence of TQSFILRT (SEQ ID NO: 14). In some examples, mosunetuzumab comprises (1) an anti-CD20 arm comprising at least one (e.g., one, two, three, or four) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 17-20, respectively, and / or at least one (e.g., one, two, three, or four) of light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 21-24, respectively, and (2) an anti-CD3 arm comprising at least one (e.g., one, two, three, or four) of heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 25-28, respectively, and / or at least one (e.g., one, two, three, or four) of light chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 29-32, respectively.In some examples, mosunetuzumab is an anti-CD2 antibody comprising: (1) (a) a VH domain comprising SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); (b) a VL domain comprising SEQ ID NO: 8, or an amino acid sequence having at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); or (c) an anti-CD2 antibody comprising a first binding domain comprising a VH domain such as in (a) and a VL domain such as in (b). and (2) an anti-CD3 arm comprising: (a) a VH domain comprising SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); (b) a VL domain comprising SEQ ID NO: 16, or an amino acid sequence having at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); or (c) a second binding domain comprising a VH domain such as in (a) and a VL domain such as in (b). In some examples, mosunetuzumab comprises (1) an anti-CD20 arm comprising a first binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8, and (2) an anti-CD3 arm comprising a second binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO:15 and a VL domain comprising the amino acid sequence of SEQ ID NO:16.

[0149] In some examples, mosunetuzumab has International Nonproprietary Name (INN) List 117 (WHO Drug Information, Vol. 31, No. 2, 2017, p. 303) or CAS Registry Number 1905409-39-3, and has (1) an anti-CD20 arm comprising the heavy chain and light chain sequences of SEQ ID NOs: 33 and 34, respectively, and (2) an anti-CD3 arm comprising the heavy chain and light chain sequences of SEQ ID NOs: 35 and 36, respectively.

[0150] In some examples, mosunetuzumab comprises: (1) (a) a heavy chain comprising SEQ ID NO: 33, or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) thereto; (b) a light chain comprising SEQ ID NO: 34, or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) thereto; (c) an anti-CD20 arm comprising a first binding domain comprising a heavy chain as in (a) and a light chain as in (b); and (2) (a (b) an anti-CD3 arm comprising a heavy chain comprising SEQ ID NO: 36, or an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to that sequence; or (c) an anti-CD3 arm comprising a second binding domain comprising a heavy chain as in (a) and a light chain as in (b). In some examples, mosunetuzumab comprises (1) an anti-CD20 arm comprising a first binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34, and (2) an anti-CD3 arm comprising a second binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36.

[0151] The amino acid sequence of mosunetuzumab is summarized in Table 1 below. [Table 1]

[0152] Mosunetuzumab can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567.

[0153] IV. Recombinant Methods and Compositions Mosunetuzumab can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid or one or more isolated nucleic acids encoding mosunetuzumab as described herein is provided. In some embodiments, such nucleic acids may encode an amino acid sequence comprising the VL and / or the VH of mosunetuzumab (e.g., the light chain and / or heavy chain of mosunetuzumab). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of mosunetuzumab and an amino acid sequence comprising the VH of mosunetuzumab, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of mosunetuzumab and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of mosunetuzumab. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell). The antibody may be produced by culturing a host cell containing a nucleic acid encoding mosunetuzumab, as described above, under conditions suitable for expression of the antibody, and optionally recovering mosunetuzumab from the host cell (or host cell culture medium).

[0154] For recombinant production of mosunetuzumab, for example, as described above, one or more nucleic acids encoding mosunetuzumab are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0155] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the present invention can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0156] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways and result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0157] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0158] Plant cell cultures may also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0159] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293T as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); TRI cells as described, for example, in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC Other useful mammalian host cell lines include DHFR 5 cells, and FS4 cells. - Examples of suitable host cell lines for antibody production include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0160] V. Assay The mosunetuzumab of the pharmaceutical composition of the present disclosure may be identified, screened, or characterized for its physical / chemical properties and / or biological activity by various assays known in the art and described herein.

[0161] 1. Binding Assay In one aspect, the mosunetuzumab of the pharmaceutical composition of the present disclosure is tested for its antigen binding activity by known methods, such as, for example, ELISA, Western blot, etc.

[0162] For example, a competitive assay can be used to identify antibodies that compete with mosunetuzumab for binding to its antigen. In an exemplary competitive assay designed to characterize binding to CD3, immobilized CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CD3, excess unbound antibody is removed and the amount of label associated with immobilized CD3 is measured. A substantial decrease in the amount of label associated with immobilized CD3 in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to CD3. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0163] 2. Activity Assay In one embodiment, an assay is provided for identifying the antibody having biological activity.Biological activity can include, for example, binding to an antigen such as CD3 (e.g., CD3 on the surface of T cells) or a peptide fragment thereof, either in vivo, in vitro, or ex vivo.In the case of mosunetuzumab, biological activity can also include, for example, effector cell activation (e.g., T cell (e.g., CD8+ and / or CD4+ T cell) activation), effector cell population expansion (i.e., increase in T cell number), target cell (e.g., CD20-positive cell) population reduction (i.e., decrease in the population of cells expressing CD20 on their cell surface), and / or target cell killing.Antibodies having such biological activity in vivo and / or in vitro are provided.

[0164] In some embodiments, antibody activity includes the ability to support B cell killing and / or cytotoxic T cell activation. In certain embodiments, mosunetuzumab is tested for such B cell killing and / or activating cytotoxic effects of T cell biological activity by any of the methods described herein, particularly by Example. In some embodiments of any of these activity assays, PBMCs may be isolated from whole blood of healthy donors by FICOLL® separation. In particular, human blood may be collected in a heparinized syringe and PBMCs may be isolated using Leucosep and FICOLL-PAQUE® Plus. Optionally, CD4+ and CD8+ T cells may be isolated with a MILTENYI® kit according to the manufacturer's instructions.

[0165] Cells may be washed with RPMI medium containing 10% FBS supplemented with GlutaMax™, penicillin, and streptomycin, and approximately 200,000 suspension cells may be added to a 96-well U-bottom plate. Cells may be cultured in RPMI 1640 supplemented with 10% FBS at 37°C in a humidified standard cell culture incubator. For the BJAB cell killing assay, 20,000 BJAB cells may be incubated with effector cells, either huPBMCs or purified T cells, at the indicated ratio per assay in the presence of various concentrations of mosunetuzumab for 24 hours. For the endogenous B cell killing assay, 200,000 huPBMCs may be incubated with various concentrations of mosunetuzumab for 24 hours.

[0166] After incubation, cells may be washed with FACS buffer (0.5% BSA, 0.05% Na azide in PBS). Cells may then be stained with FACS buffer, washed with FACS buffer, and suspended in 100 μl of FACS buffer containing 1 μg / ml propidium iodide. Data may be collected on a FACSCalibur™ flow cytometer and analyzed using FLOWJO®. Viable B cells may be gated out by FACS as PI-CD19+ or PI-CD20+ B cells, and FITC beads may be added to the reaction mixture to obtain absolute cell counts as an internal counting control. The percentage of cell death may be calculated based on non-mostuzumab-treated controls. Activated T cells may be detected by CD69 and CD25 surface expression using anti-CD69-FITC and anti-CD25-PE.

[0167] 3. Stability Assay Suitable assays for measuring the stability of pharmaceutical compositions (e.g., mosunetuzumab) are known in the art and are described herein. For example, pharmaceutical compositions can be analyzed by assessing aggregate formation (e.g., by using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing reactive oxygen species (ROS) formation (e.g., by using a mild stress assay or a 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues of proteins (e.g., Met residues in antibodies (e.g., mosunetuzumab)); assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; reduced polypeptide analysis; Instability can be assessed qualitatively and / or quantitatively in a variety of different ways, including SDS-PAGE analysis to compare the peptide with the intact polypeptide; (e.g., antibody); peptide map (e.g., trypsin or LYS-C) analysis; assessment of the protein's biological activity or target binding function (e.g., antibody binding to its antigen), etc. Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, and differential glycosylation, etc. Exemplary assays are described in more detail below.

[0168] VI. Treatment Methods and Uses The pharmaceutical compositions described herein (i.e., comprising mosunetuzumab) can be formulated for use as medicaments for treating cancer. Accordingly, the present disclosure features methods including administering the pharmaceutical composition to a subject in need thereof, e.g., a subject with cancer. The pharmaceutical compositions of the present disclosure can be used to treat or delay the progression of cancer in a subject in need thereof (e.g., a human subject in need thereof), or to enhance immune function in a subject with cancer.

[0169] Mosunetuzumab binds to a CD3 molecule located on an immune effector cell and a CD20 molecule located on a target cell (e.g., a CD20-positive cell) other than an immune effector cell (e.g., a CD20 molecule located on (e.g., expressed by) a target cell (e.g., a CD20-positive cell), such as a B cell. In some embodiments, mosunetuzumab activates immune effector cells after binding to the CD3 molecule and CD20. Once activated, the immune effector cells can exert a cytotoxic and / or apoptotic effect on target cells (e.g., CD20-positive cells).

[0170] In some embodiments, the cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, the NHL is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, Burkitt-like lymphoma with 11q aberration, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (DLBCL), germinal center B-cell-like (GCB) diffuse large B-cell lymphoma ( DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, and HHV8-associated multicentric Castleman disease Originates from large B-cell lymphoma, B-cell leukemia, follicular lymphoma (FL), in situ follicular neoplasia, mantle cell lymphoma (MCL), in situ mantle cell neoplasia, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic Selected from the group consisting of leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma, childhood follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, and primary effusion lymphoma.In certain embodiments, the cancer is germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), or Burkitt's lymphoma (BL).

[0171] In some embodiments, the NHL is DLBCL, GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL, or BL. In some embodiments, the NHL is FL or DLBCL. In some embodiments, the NHL is relapsed and / or refractory (R / R). In some embodiments, the NHL is R / R NHL. In some embodiments, the R / R FL has relapsed after or is refractory to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) prior systemic therapies. In some embodiments, the preceding one or more systemic therapies include an anti-CD20 monoclonal antibody. In some embodiments, the prior systemic therapy or multiple systemic therapies include an alkylating agent (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, altretamine, or thiotepa). In certain embodiments, the prior systemic therapy or multiple systemic therapies include both an anti-CD20 monoclonal antibody and an alkylating agent.

[0172] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and glioblastoma.

[0173] Mosunetuzumab is administered in a dose of about 0.1 mg to about 100 mg (e.g., 0.1 mg to 80 mg, 0.5 to 70 mg, 1 mg to 60 mg, 0.1 mg to 2 mg, 0.5 mg to 1.5 mg, 1 mg to 5 mg, 1.5 mg to 2.5 mg, 1 mg to 30 mg, 15 mg to 45 mg, 5 mg to 10 mg, 10 mg to 15 mg, 20 mg to 40 mg, 20 mg to 30 mg, 30 mg to 40 mg, 25 mg to 35 mg, 50 mg to 100 mg, 50 mg to 60 mg, 55 mg to 65 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 mg). The compound may be formulated for administration to a subject at a dose of about 1 mg to about 100 mg, e.g., about 0.5 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 13.5 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, or about 100 mg. In some embodiments, the method comprises administering mosunetuzumab to a subject at a dose of about 1 mg to about 60 mg. In certain embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, about 2 mg, about 6 mg, about 9 mg, about 13.5 mg, about 20 mg, about 30 mg, or about 60 mg. In some embodiments, the method comprises administering mosunetuzumab to the subject at a dose of about 1 mg, 2 mg, 30 mg, or 60 mg.

[0174] In some embodiments, the pharmaceutical composition is diluted with saline before administration to a subject. In some embodiments, the saline is normal saline. In some embodiments, the saline contains sodium chloride (NaCl). In some embodiments, the saline contains 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl. In certain embodiments, the saline contains 0.45% or 0.9% (w / v) NaCl. In some embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml to about 0.3 mg / ml (e.g., about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.75 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, In certain embodiments, after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0175] In one embodiment, a pharmaceutical composition disclosed herein (e.g., comprising mosunetuzumab) is administered to a subject in a dosing regimen comprising at least three 21-day (±3 day) dosing cycles, wherein (a) the first 21-day dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of mosunetuzumab administered to the subject on or about days 1, 8 (±1 day), and 15 (±1 day) of the first dosing cycle, respectively, wherein C1D1 is administered in an amount of about 1 (b) the second administration cycle comprises a single dose (C2D1) of mosunetuzumab administered to the subject on or about day 1 of the second administration cycle, wherein C2D1 is about 60(±5) mg; (c) the third administration cycle comprises a single dose (C3D1) of mosunetuzumab administered to the subject on or about day 1 of the third administration cycle, wherein C3D1 is about 30(±3) mg. In some embodiments, the administration regimen comprises 1 to 14 additional administration cycles, each comprising an additional single dose of about 30(±0.5) mg of mosunetuzumab. In some embodiments, the administration regimen comprises 1 to 5 (e.g., 1, 2, 3, 4, or 5) additional administration cycles. In certain embodiments, the administration regimen comprises 5 additional administration cycles. In some embodiments, the subject is administered each additional single dose of mosunetuzumab on or about day 1 of each additional administration cycle.

[0176] The present disclosure also provides methods for co-administration of a pharmaceutical composition (i.e., comprising mosunetuzumab) with at least one additional therapeutic agent (e.g., one, two, three, four, or more additional therapeutic agents). For example, the additional therapeutic agent can include a PD-1 axis binding antagonist, such as a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. PD-L1 binding antagonists useful in the present methods and pharmaceutical compositions include, for example, atezolizumab (MPDL3280A), MDX-1105 (BMS-936559), and MEDI4736 (durvalumab). For example, in some specific examples, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874 and WO 2016 / 201425. MEDI4736 (durvalumab) is an anti-PD-L1 monoclonal antibody described in WO 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559. Examples of anti-PD-L1 antibodies useful in the methods of the disclosure, and methods for making them, are described in PCT Publication Nos. WO 2010 / 077634, WO 2007 / 005874, and WO 2011 / 066389, as well as U.S. Patent No. 8,217,149 and U.S. Patent Application Publication No. 2013 / 034559.

[0177] PD-1 binding antagonists include anti-PD-1 antibodies, such as those selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, AMG404, REGN2810 (cemiplimab; LIBTAYO®), and BGB-108. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO 2006 / 121168. MK-3475, also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in WO 2009 / 114335. In another example, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In another example, the PD-1 binding antagonist is REGN2810 (LIBTAYO®), also known as cemiplimab. In another example, the PD-1 binding antagonist is AMP-224, B7-DCFc, or PD-L2Fc fusion protein, as described in WO 2017 / 058780.

[0178] PD-L2 binding antagonists include, for example, antibodies (e.g., anti-PD-L2 antibodies) and immunoadhesins. In some embodiments, the additional therapeutic agent includes obinutuzumab (an anti-CD20 antibody), rituximab (an anti-CD20 antibody), an antibody-drug conjugate (ADC), a corticosteroid, or tocilizumab (an anti-IL-6R antibody).

[0179] For example, an additional therapeutic agent useful for co-administration can be an ADC, such as an anti-CD79b ADC (e.g., polatuzumab vedotin; see, e.g., WHO Drug Information (2012) vol. 26, No. 4, 437-438). When the methods described herein include combination therapy, such as the specific combination therapy described above, the combination therapy encompasses simultaneous administration of a pharmaceutical composition (i.e., comprising mosunetuzumab) with one or more additional therapeutic agents, and such simultaneous administration may be combined or separate. Furthermore, the two or more therapeutic agents may be formulated together or separately. When the two or more therapeutic agents are administered separately, administration of the pharmaceutical composition (i.e., comprising mosunetuzumab) may occur before, simultaneously with, and / or after administration of the one or more additional therapeutic agents.

[0180] The pharmaceutical compositions of the present disclosure may be administered, for example, intravenously. In certain embodiments, mosunetuzumab is administered intravenously. In particular, the mosunetuzumab drug product is diluted with saline (e.g., normal saline) prior to administration. In some embodiments, the saline contains sodium chloride (NaCl). In some embodiments, the saline contains 0.1-1.5% (e.g., 0.1-1.2%, 0.3-1.5%, 0.4-0.5%, 0.3-1%, 0.8-1%, 0.85-0.95%, e.g., about 0.1%, about 0.3%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, or about 1.2%) (w / v) NaCl. For example, using a 1 mg / ml formulation of mosunetuzumab, the following dilutions may be used to deliver the indicated doses (Table 2): [Table 2]

[0181] In one embodiment, the administration of mosunetuzumab and the administration of the additional therapeutic agent may occur within about 1 month, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0182] The methods described herein may result in an improved benefit-risk profile for patients with cancer who are being treated with mosunetuzumab. In some examples, treatment using the methods described herein, in which mosunetuzumab is administered in the context of a fractionated, ascending-dose dosing regimen, may result in a lower incidence of undesirable events, such as cytokine-driven toxicities (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe leukemia, and the like, following treatment with mosunetuzumab using the fractionated, ascending-dose dosing regimen disclosed herein, compared to treatment with mosunetuzumab using a non-fractionated dosing regimen. may result in a reduction (e.g., 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) or complete inhibition (100% reduction) of tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity.

[0183] For all methods and pharmaceutical preparations described herein, mosunetuzumab is formulated, dosed, and administered in accordance with good medical practice.Factors to consider in this regard include the specific cancer being treated, the specific mammal being treated, the clinical condition of each patient, the cause of the cancer, the drug delivery site, administration method, administration schedule, and other factors known to medical professionals.Mosunetuzumab does not necessarily have to be one or more drugs currently used to prevent or treat the cancer in question, but it may be formulated with them.The effective amount of such other drugs depends on the amount of mosunetuzumab present in the formulation, the type of cancer or treatment, and other factors mentioned above.Mosunetuzumab can be appropriately administered to patients over a course of treatment.

[0184] VII. Manufactured articles In another aspect of the present disclosure, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned cancers is provided. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a pharmaceutical composition used alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is mosunetuzumab, as described herein. The label or package insert indicates that the composition is used to treat a selected condition (e.g., cancer) and further includes information related to at least one of the dosing regimens described herein.

[0185] The pharmaceutical composition may be supplied in a container having a volume of 1 ml to 100 ml (e.g., 1 ml to 5 ml, 5 ml to 10 ml, 10 ml to 15 ml, 15 ml to 20 ml, 20 ml to 25 ml, 25 ml to 30 ml, 30 ml to 40 ml, 40 ml to 50 ml, 50 ml to 60 ml, 60 ml to 70 ml, 70 ml to 80 ml, 80 ml to 90 ml, or 90 ml to 100 ml, for example, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml, about 90 ml, or about 100 ml). In certain embodiments, the container has a volume of about 50 ml (e.g., about 40 ml, about 45 ml, about 46 ml, about 47 ml, about 48 ml, about 49 ml, about 50 ml, about 51 ml, about 52 ml, about 53 ml, about 54 ml, about 55 ml, or about 60 ml). In another specific embodiment, the container has a volume of about 2 ml (e.g., about 1 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, about 2 ml, about 2.1 ml, about 2.2 ml, about 2.3 ml, about 2.4 ml, about 2.5 ml, or about 3 ml).

[0186] In some embodiments, the container is a stainless steel or nickel alloy container (e.g., HASTELLOY®), such as a tank, mini-tank, canister, or can. In some examples, the pharmaceutical composition in such a container is a DS and may be further diluted, for example, into a DP (e.g., in final vial configuration) before use. Alternatively, the pharmaceutical composition in the container is a DP. In some embodiments, if the DP is intended to be administered with a diluent (e.g., saline; e.g., saline containing 0.45% or 0.9% (w / v) NaCl) or in combination with other therapeutic agents, the DP may be at a higher concentration than that administered to a subject. In some embodiments, the DP is contained in a container such as an IV bag or syringe (e.g., for delivery via a syringe pump). In some embodiments, the DP may be diluted in an IV bag using a diluent (e.g., saline; e.g., saline; e.g., saline containing 0.45% or 0.9% (w / v) NaCl).

[0187] In some embodiments, the article of manufacture includes a vial (e.g., a glass vial) having a volume of about 1 ml or more, e.g., about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about 19 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 50 ml, or more. In certain embodiments, the vial has a volume of about 50 ml (e.g., about 40 ml, about 45 ml, about 46 ml, about 47 ml, about 48 ml, about 49 ml, about 50 ml, about 51 ml, about 52 ml, about 53 ml, about 54 ml, about 55 ml, or about 60 ml). In another specific embodiment, the vial has a volume of about 2 ml (e.g., about 1 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, about 2 ml, about 2.1 ml, about 2.2 ml, about 2.3 ml, about 2.4 ml, about 2.5 ml, or about 3 ml). In some embodiments, the vial is disposable. In some embodiments, the vial contains about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 30 mg, about 45 mg, about 60 mg, or more of mosunetuzumab. In some embodiments, the vial contains a pharmaceutical composition comprising mosunetuzumab, polysorbate 20 (PS20), methionine, a buffering agent, and a carrier, wherein the molar ratio of PS20 to mosunetuzumab is 100 or less, the concentration of PS20 is 0.01% to 0.1% weight / volume (w / v), the concentration of methionine is 1 mM to 50 mM, and the concentration of the buffering agent is 5 mM to 20 mM. In some embodiments, the container closure system comprises one or more, or all, of a glass vial, a stopper, and a cap.

[0188] Additionally, the article of manufacture may include (a) a first container containing a composition, the pharmaceutical composition comprising mosunetuzumab as described herein, and (b) a second container containing a pharmaceutical composition, the pharmaceutical composition comprising an additional cytotoxic or other therapeutic agent. Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextrose solution, etc. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents (e.g., saline; e.g., saline containing 0.45% or 0.9% (w / v) NaCl), filters, needles, and syringes. [Example]

[0189] VIII. Working Examples The following are examples of methods and compositions of the present disclosure. It will be understood that various other embodiments may be practiced given the general description provided above, and these examples are not intended to limit the scope of the claims.

[0190] Example 1. Drug Development This example demonstrates how to formulate and use mosunetuzumab (BTCT4465A), for which researchers observed that a relatively low protein concentration and a relatively high surfactant concentration appear to be preferable in the formulation to ensure accurate delivery of low doses of mosunetuzumab.

[0191] Mosunetuzumab is based on the human IgG1 isotype and is intended for the treatment of B-cell malignancies by recruiting and activating T cells. The CD20 arm of mosunetuzumab is directed against the B-lymphocyte antigen CD20, a glycosylated phosphoprotein expressed on the surface of B cells. The CD3 arm binds to and recruits T cells, which are activated upon target engagement with CD20, resulting in robust T-cell proliferation and cell killing.

[0192] The amino acid sequence comprising mosunetuzumab is summarized in Table 1 above.

[0193] We produced recombinant mosunetuzumab in two separate CHO cell lines using knob-in-hole technology, including the glycosylation mutation N297G to reduce or eliminate ADCC function. Mosunetuzumab is composed of one anti-CD20 half-antibody with a knob (anti-CD20 arm) and one anti-CD3 half-antibody with a hole (anti-CD3 arm), assembled using glutathione reduction. Mosunetuzumab was active against indolent (e.g., non-dividing) and chemoresistant cells, and a prior immune response to the tumor was not a prerequisite for activity.

[0194] Dose ranges in subsequent and ongoing clinical trials varied due to the use of a stepped, split-dose scheme in which, during cycle 1, patients received a low dose (e.g., 1 mg) on ​​day 1, an intermediate dose (e.g., 2 mg) on ​​day 8, and the full dose (e.g., 20 mg or 30 mg) on ​​day 15. When combining protein concentration with low or high surfactant levels, several formulation options were considered (Figure 1). In particular, relatively low protein concentrations and relatively high surfactant concentrations were considered preferable to ensure accurate delivery of low doses using intravenous fluid (IV) bags, reduce or avoid the use of diluents, allow for dosing flexibility, and reduce or eliminate waste.

[0195] Example 2. Knowledge building test A knowledge-building study was performed after early-stage development to evaluate the impact of formulation parameters on formulation stability and identify key risks for the formulation. Importantly, the results of this study suggested that oxidative degradation (e.g., oxidative degradation associated with other forms of degradation, such as aggregation, fragmentation, and surfactant degradation) was a major risk factor for the Phase III clinical DP formulation (i.e., low-concentration mosunetuzumab DP formulation). Furthermore, the results of this study suggested that low-protein mosunetuzumab formulations, particularly those with high histidine, sucrose, and PS20 concentrations, may be susceptible to oxidative degradation. Furthermore, the results of this study suggested that the addition of methionine (L-methionine; L-Met) inhibited oxidative degradation, thereby reducing the risk of other forms of degradation (e.g., aggregation, fragmentation, and / or surfactant degradation).

[0196] Example 3. Formulation Development The objective of the study in this example was to characterize the risks associated with pharmaceutical compositions containing mosunetuzumab and identify formulation parameters that reduce these risks. The study summarizes the effects of various components of DS and DP formulations and the containers in which they may be stored. The study also found that a key feature of such formulations is the molar ratio of surfactant to mosunetuzumab, with the protein present at a low molar concentration and the surfactant at a higher molar concentration.

[0197] 3.1.IV Bag Agitation Test To determine the appropriate amount and composition of surfactant for use in a diluent-free formulation delivered via an IV bag to support a wide dose range, an IV bag agitation study was conducted. Several factors were characterized, including protein concentration, dose, IV bag size, surfactant composition, and surfactant concentration.

[0198] Mosunetuzumab was formulated at various protein concentrations (1 mg / ml, 5 mg / ml, 10 mg / ml) in 20 mM histidine acetate (HisOAc), 240 mM sucrose, pH 5.8. A range of doses (1 mg, 2 mg, 5 mg, 20 mg, and 30 mg) was tested, and 50 ml and 100 ml polyoxyethylene (PO) bags were evaluated. 50 ml and 100 ml PO IV bags have previously been determined to be high-risk IV bags due to protein aggregation caused by their relatively large headspace and high wall rigidity compared to polyvinyl chloride (PVC) IV bags of the corresponding size. Three surfactants were evaluated: polysorbate 20 (PS20), poloxamer 188 (P188), and ultra-purified polysorbate 20 (srPS20). PS20 and P188 were tested over the full dose range, and limited testing was performed with srPS20 to allow for comparability with PS20 in agitation studies. SrPS20 was then discontinued, and testing was simplified.

[0199] In these experiments, DP surfactant concentrations were varied to determine the minimum surfactant concentration in the IV bag required to prevent aggregation and particle formation during IV bag agitation stress. For each test condition, mosunetuzumab DP was infused into the IV bag and shaken at 100 rpm on an orbital shaker at 2-8°C for up to 24 hours. Samples were taken at 0, 1, 6, and 24 hours and evaluated for subvisible particles by high-precision particle counting (HIAC) and for soluble aggregates by size-exclusion high-performance liquid chromatography (SE-HPLC). Conditions were considered passed if there was no substantial increase in soluble aggregate or particle count. The increase in soluble aggregates was assessed by the observed qualitative change in the SE-HPLC chromatogram compared to time 0. Particle formation was observed as a significant increase in the number of subvisible particles compared to time 0 (an increase of more than 1,000 cumulative particles ≥ 2 μm / ml).

[0200] Data from this study was analyzed to determine the minimum amount of surfactant in an IV bag required to support Phase III and diluent-free commercial dose administration. Figure 2 shows how DP protein concentration and dose together affected the minimum required PS20 concentration in a 100 ml PO IV bag. Similar results were observed for 50 ml PO IV bags. A relatively high ratio of PS20 concentration to protein concentration (e.g., at low doses, e.g., 1 mg / ml) ensured sufficient surfactant levels in the final dose solution in the IV bag. These results suggest that a higher ratio of PS20 to protein concentration may be better for ensuring protein stability at low DP doses (e.g., 1 mg / ml), for example, in an IV bag.

[0201] The appropriate minimum surfactant level was determined for each surfactant composition at 1 mg / ml DP, which was identified as the effective protein concentration supporting low-dose administration, as shown in Figure 3. A minimum of 0.05% (w / v) PS20 (surfactant to mosnetuzumab molar ratio of approximately 59) and super-refined PS20 (srPS20), and a minimum of 0.08% (w / v) P188 (surfactant to mosnetuzumab molar ratio of approximately 14) were determined to be necessary to prevent aggregation and particle formation during the shaking stress conditions described above. To support manufacturing range, the PS20 target level was set at 0.06% (w / v) (surfactant to mosnetuzumab molar ratio of approximately 71) and the P188 target level was set at 0.10% (w / v) (surfactant to mosnetuzumab molar ratio of approximately 17) for the following DP formulation development.

[0202] 3.2. Effect of surfactants on product quality The product quality impact (PQI) of various surfactant types was evaluated in a stability study. The oxidation risk of mosunetuzumab was of particular interest due to the oxidative effects observed in the knowledge-building study (Example 2). Furthermore, PS20 is known to be susceptible to oxidation, and high levels of PS20 (e.g., 0.06%, as selected by IV bag agitation testing) may increase the risk of protein oxidation. In this study, protein concentrations (60 mg / ml, 10 mg / ml, 1 mg / ml) and surfactant compositions (PS20, srPS20, P188) were evaluated in a full factorial study. All materials were formulated at their respective protein concentrations using 20 mM histidine acetate (HisOAc), 240 mM sucrose, and 0.1% (w / v) surfactant at pH 5.8. For PS20 or srPS20 as surfactants with protein concentrations of 60 mg / ml, 10 mg / ml, and 1 mg / ml, the molar ratios of surfactant to mosunetuzumab were approximately 2, 12, and 119, respectively. For P188 as surfactants with protein concentrations of 60 mg / ml, 10 mg / ml, and 1 mg / ml, the molar ratios of surfactant to mosunetuzumab were approximately 0.3, 1.7, and 17, respectively. Samples were hand-filled into 6 ml glass vials with 3 ml fill volumes, stoppered using standard components, capped, and stored at 40 °C for up to 1 month and at 30 °C for up to 3 months. Assays included peptide mapping to quantify oxidation, HIAC to quantify aggregation and particle formation, evaporative light scattering detector (ELSD) to quantify surfactant degradation, and SE-HPLC to quantify size heterogeneity.

[0203] In compositions containing PS20 or P188, oxidation of Met257 (M257) increased only slightly as the protein concentration decreased from 60 mg / ml to 1 mg / ml (Figure 4A and Figure 4B). However, with srPS20, oxidation increased significantly at a protein concentration of 1 mg / ml (Figure 4C). Increased fragmentation and aggregation, as well as loss of surfactant, were also observed in samples with 1 mg / ml srPS20. Based on these results, srPS20 was ruled out as a surfactant of choice for mosunetuzumab formulations.

[0204] 3.3. Histidine Concentration, Ambient Light Exposure, and Antioxidant Screening Histidine is susceptible to oxidation, and its presence in formulation buffers can induce protein oxidation. In this example, the effect of histidine concentration was further characterized. The knowledge-building tests described in Example 2 and the tests using the above surfactant types revealed that mosunetuzumab is susceptible to oxidation from heat stress. However, the effect of light stress on mosunetuzumab has not been characterized. Here, the susceptibility of tryptophan residues in mosunetuzumab to oxidation was quantified.

[0205] Two formulations were evaluated for heat stress, and four formulations were evaluated for light stress (Table 3). All formulations contained 1 mg / ml mosunetuzumab, 160 mM sucrose, 0.04% (w / v) PS20, pH 5.5, in addition to the specified composition. In this example, the molar ratio of mosunetuzumab to surfactant was approximately 48. Formulation 7 (F7) was identified in a knowledge-building study as being at risk for high oxidation due to heat stress. A low-histidine F7 formulation (F7-LowHis) was included in this study to evaluate the impact of reduced histidine concentration. In a mild stress study, F7 was supplemented with the antioxidant methionine, either alone or in combination with N-acetyltryptophan (NAT), to evaluate the antioxidant's effect on mild stress-mediated degradation. Formulations were filled into 6 ml vials at 3 ml fill volumes, stoppered, capped, and subjected to heat stress (up to 1 month at 40 °C and up to 3 months at 25 °C) and ambient light stress (300,000 lux-hrs at 25 °C). Ambient light exposure was performed by incubating samples for 54 hours in a fluorescent light box maintained at room temperature and 5500 lux light intensity. Assays included peptide mapping to quantify oxidation, HIAC to quantify aggregation and particle formation, ELSD to quantify surfactant degradation, and SE-HPLC to quantify size heterogeneity (e.g., as a result of fragmentation and / or aggregation). [Table 3]

[0206] Compositions containing various levels of histidine (F7 and F7-lowHis) were compared under heat stress conditions at 40°C. Both samples did not contain antioxidants. As shown in Figure 5, the 30 mM histidine composition showed approximately 6% M257 oxidation after 2 weeks at 40°C, while the 10 mM histidine composition showed less than 1% M257 oxidation. Furthermore, aggregation levels of both dimers and high molecular weight species (HMWS) were also higher in the 30 mM histidine sample. The results suggest that higher surfactant levels and lower histidine acetate (HisOAc) concentrations may be better at preventing oxidative stress under heat stress conditions.

[0207] The effects of histidine concentration and antioxidants were evaluated under ambient light exposure, a sensitive type of stress that can induce oxidation. As shown in Figure 6, no substantial changes in oxidation were observed at either site (Met or Trp) in any of the samples tested, indicating that F7 is stable under the relevant ambient light stress, despite its high risk of oxidation. NAT did not provide any additional benefit and was therefore not included in subsequent formulation development.

[0208] 3.4. Optimization of methionine concentration Hydrogen peroxide addition studies were performed to identify the preferred concentration of methionine, which was identified as a critical excipient. Samples (control formulations) containing 1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, and 160 mM sucrose at pH 5.8 were prepared with or without hydrogen peroxide (HO) and various levels of Met (Table 4). In these samples, the molar ratio of surfactant pair to mosunetuzumab is approximately 95. The control formulation contains a relatively high concentration of histidine, a relatively high concentration of PS20, and a relatively low concentration of sucrose, which increases the risk of oxidation.

[0209] Hydrogen peroxide (2,000 ng / ml) was used as an oxidative stress challenge for antioxidant protection. Samples were filled into 20 cc vials with a 3 ml fill volume to represent container conditions with a high risk of oxidation due to the large headspace. The vials were stoppered, capped, and placed at 40°C for 1 month, 25°C for 6 months, or for real-time stability, i.e., thermal stability, at 2–8°C. For ambient light testing, samples were subjected to ambient light exposure at room temperature for up to 300,000 lux-hours (5,500 lux for 54 hours). Samples were obtained from the ambient light test at 0, 24, 48, and 54 hours, including a dark control covered with aluminum foil. Hydrogen peroxide concentrations were measured using the AMPLEX® Hydrogen Peroxide / Peroxidase Assay (ThermoFisher Scientific; Waltham, MA). Assays used to monitor product quality (e.g., stability) included color, pH, turbidity (by ultraviolet spectroscopy (UV-spec)), strength (by UV-spec), charge variants (by imaged capillary isoelectric focusing (icIEF) and microchip sieving electrophoresis (MCE-SDS)), size heterogeneity (by SE-HPLC), visible and subvisible particles (by HIAC), potency, oxidation (by peptide mapping), methionine concentration (by mass spectrometry), and polysorbate concentration (by ELSD). [Table 4] a All samples contained 1 mg / ml mosunetuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8, in addition to the composition specified in the table.

[0210] After 12 months of storage at 2-8°C (e.g., 5°C), the HO present in the formulation without Met was completely depleted (Figure 7A). At 12 months, 41.5% M257 oxidation was observed, in contrast to 3.7% M257 oxidation in the control sample (Figure 7B). When a minimum of 2.5 mM Met was added to the formulation, HO was completely consumed after 1 month (Figure 7A), and M257 oxidation remained comparable to the control sample throughout the study period (Figure 7B). No differences were observed between formulations containing 2.5 mM, 5 mM, and 10 mM Met. No changes in other properties were observed.

[0211] Under accelerated thermal conditions (i.e., 25°C), HO levels in Met-free samples decreased to 130 ng / ml at 2 months and then accumulated back to levels of 1,000 ng / ml by 6 months (Figure 8A). Control samples also showed an increase in HO after 2 months (Figure 8A). This observation is consistent with a slow phase of increased Met oxidation, represented by M257, in the first 2 months for Met-free spiked samples and a fast phase of Met oxidation in the later portion up to 6 months for both Met-free spiked samples and controls (Figure 8C). This observation is also consistent with an increase in tryptophan 107 (i.e., Trp107 or W107) oxidation in the CD20 arm (Figure 8B), protein aggregation (Figure 8D), and fragmentation (Figure 8E) between 2 and 6 months for both Met-free spiked samples and control samples. This observed HO production may be related to oxidative PS20 degradation in both the Met-free control and spiked samples, which may lead to further protein degradation. In contrast, upon addition of 2.5–10 mM Met, HO was completely consumed after 1 week and did not re-accumulate over the course of 6 months of incubation (Figure 8A). Increases in M257 oxidation, W107 oxidation, aggregation, fragmentation, and PS20 degradation were significantly reduced by 2.5 mM Met throughout 6 months of incubation and completely inhibited by 5 mM and 10 mM Met (Figure 8B–E), suggesting that 5 mM Met is the minimum antioxidant requirement for this formulation under these conditions. A similar trend was observed from stress stability results at 40 °C on shorter timescales.

[0212] All samples in Table 5 were also subjected to ambient light stress (up to 300,000 lux-hours) at 25° C. Consistent with the histidine concentration studies, ambient light did not cause extensive oxidation, and the small amount of M257 oxidation produced in samples containing no Met was completely inhibited by the addition of 2.5 mM Met. [Table 5] a All formulations contained 1 mg / ml mosunetuzumab.

[0213] 3.5 Screening of DP formulations Based on the information obtained from the above studies, a DP formulation screening study was conducted. Target formulations and high-risk oxidation (high His, high PS20, low sucrose) were evaluated at various pH and Met concentrations. Each formulation was sterile filtered and filled into 15 ml glass vials with a 5 ml fill volume. The vials were stoppered with 20 mm Daikyo D777-1 liquid stoppers (Okagami, Chiba, Japan), capped with aluminum flip-top caps, and stored upright for stability according to the program described in Table 6. The assay was configured to monitor color, pH, turbidity (by UV-spec), strength (by UV-spec), charge variants (by icIEF and mCE-SDS), size heterogeneity (by SE-HPLC), visible and subvisible particles (by HIAC), potency, oxidation (by peptide mapping), methionine concentration (by mass spectrometry), and polysorbate concentration (by ELSD). [Table 6] X = 3 vials; X* = 5 vials; D = day, W = week, M = month

[0214] Time zero (T0) characterization of the formulations is summarized in Table 7. After 12 months at 5°C, no changes were observed for any of the formulations (Table 8). After 24 months of storage at 5°C, consistent changes were observed across all five formulations by iCIEF and CE-SDS. There was a slight increase in the proportion of acidic variants by icIEF, and the pre-peak CE-SDS results appear to show a slight increase after 24 months at 5°C, but the same increase was observed across all formulations. No changes were observed in other assays.

[0215] After 6 months of storage at 25°C and 60% relative humidity (RH; accelerated conditions) (Table 9), similar changes were observed in all five formulations for SE-HPLC main peak loss (0.3-0.4%), icIEF acidity increase and main peak loss (12.6-17%), and mCE-SDS main peak loss and pre-peak increase (1.0%). No changes were observed when other assays were performed.

[0216] After 1 month of storage at 40 °C and 75% RH (stress conditions) (Table 10), similar changes were observed in all five formulations for SE-HPLC main peak decrease (0.4-0.6%, Figures 9A-C), icIEF acidity increase and main peak decrease (17.1-19.8%, Figures 10A-C), and mCE-SDS main peak decrease and pre-peak increase (1.4-1.9%, Figures 11A and 11B). No changes were observed in other assays performed.

[0217] The results of this study showed that mosunetuzumab DP is stable within the pH range of 5.5 to 6.1, even under high-risk conditions for oxidation (high L-histidine, low sucrose, low L-methionine, high polysorbate). [Table 7] [Table 8] TIFF0007808151000010.tif250170TIFF0007808151000011.tif253170TIFF0007808151000012.tif253170 [Table 9] TIFF0007808151000014.tif253170TIFF0007808151000015.tif253170TIFF0007808151000016.tif253170 [Table 10] TIFF0007808151000018.tif248170TIFF0007808151000019.tif253170TIFF0007808151000020.tif253170

[0218] 3.6 Screening of DS formulations DS stability was evaluated in stainless steel minicans. Samples included the target formulation (Formulation 1), Formulation 2, and Formulation 3, with the compositions shown in Table 11. All formulations contained 10 mg / ml mosunetuzumab and 0.06% (w / v) PS20. In these examples, the molar ratio of surfactant to mosunetuzumab is approximately 7. Formulations 2 and 3 are high-risk formulations due to oxidation at relatively low and high pH, ​​respectively. To quantify the impact of headspace, the risk of accelerated protein oxidation by metal eluates was assessed by incubating DS in surface-mounted minicans with various fill volumes. [Table 11]

[0219] Each formulation was sterile filled into 25 ml 316L stainless steel mini-cans with a 15 ml fill volume. The mini-cans were stored upright under real-time (-20°C), accelerated (2°C to -8°C), stress (25°C, 60% relative humidity; RH) conditions, and optionally stable at -40°C. The mini-cans were stored under frozen conditions, undergoing no more than three freeze-thaw cycles. To control for variability within the cans, two cans were placed in most temperature conditions.

[0220] To test for protein oxidation induced by metal leachates, a total of four 45 ml mini-cans that showed surface wear (pitting, scratches, etc.) were selected and filled with a low pH formulation (pH 5.5) that was considered to pose a high risk of protein oxidation. These mini-cans were placed in a stable condition at 2-8 °C and 25 °C at 60% RH (two mini-cans at each temperature). To create variability in headspace, the DS fill volume of the mini-cans was varied between a high fill volume (40 ml) and a low fill volume (15 ml).

[0221] Assays were performed to observe pH, oxidation (by peptide mapping), size variation (by SE-HPLC), charge variants (by mCE-SDS and iCIEF), color and appearance, subvisible particles (by HIAC), visible particles, concentration (by UV-SpecScan), turbidity, potency, and PS20 concentration (by ELSD). No changes were observed for any formulation tested after 12 months of storage at -40°C or -20°C, and no changes were observed for any formulation tested after 6 months of storage at 5°C. High- and low-load samples in surface-abraded 45ml mini-cans showed no differences compared to samples stored in 25ml mini-cans at 5°C.

[0222] In summary, mozunetuzumab DS was stable within pH 5.5-6.1, even in high-risk formulations due to oxidative conditions (high His and low Met for oxidation, low sucrose for freeze stability) where metal elution-induced oxidation was difficult (low fill volume of surface-mounted minicans). Thus, these data indicate that such compositions (e.g., compositions with high His, low Met, and low sucrose) can be used at protein levels up to approximately 10 mg / ml as needed in some DS formulations.

[0223] 3.7 Confirmatory Stability Testing of DS Formulations (Additional Formulations) Additional DS formulations were tested to assess whether low histidine and high Met concentrations affect DS stability. Stability had not previously been tested for histidine acetate concentrations below the target of 10 mM or methionine concentrations above 10 mM. The formulations are shown in Table 12 below. All formulations contained 10 mg / ml mosunetuzumab and 0.06% (w / v) PS20. In these formulations, the molar ratio of surfactant to mosunetuzumab is approximately 7. [Table 12]

[0224] Each formulation was sterile filtered and filled into 25 ml 316L stainless steel mini-cans with a 16 ml fill volume. The mini-cans were stored upright in stable conditions at accelerated (2-8°C) and stressed (25°C / 60% RH) temperatures. Two cans were placed in a stable condition to control for mini-can variability.

[0225] Assays were performed to test pH, oxidation (by peptide mapping), methionine concentration, potency, size variants (by SE-HPLC), charge variants (by mCE-SDS and iCIEF), color and appearance, visible particles, concentration (by UV-SpecScan), turbidity, and PS20 concentration (ELSD).

[0226] No changes were observed for any of the formulations tested after one month of storage at 5°C. The high-methionine and low-histidine formulations showed no differences compared to the target sample. After one month of storage at 25°C, consistent changes were observed with both DS formulations, with a slight increase in LMWS by SE-HPLC (0.2%) and CE-SDS, and a decrease in the main peak and an increase in the acidic peak by icIEF. No changes were observed in other assays. This study demonstrated that mosunetuzumab DS is stable within a methionine concentration range of 10-15 mM and a histidine acetate concentration range of 5-10 mM.

[0227] 3.8 DS Freeze-Thaw Stability The freeze-thaw stability of DS was evaluated by stressing each formulation identified in Table 11 with seven freeze-thaw cycles in mini-cans. 25 ml 316L stainless steel mini-cans were filled with 16 ml of sterile-filtered material. One mini-can was prepared for each formulation. The freeze-thaw cycle consisted of 2 hours at -40°C, followed by at least 8 hours at -20°C, followed by storage at room temperature until the sample was completely thawed (approximately 2-3 hours). Prior to sampling, the material was mixed by gently inverting the can approximately 10 times. After the third, fourth, fifth, and seventh cycles, 2.5 ml of each mini-can was aseptically sampled.

[0228] Assays were performed for subvisible particles (HIAC), visible particles, turbidity (UVspec), oxidation (peptide map), and sizing (SE-HPLC). No changes were observed, as shown in Table 13. These data confirmed that DS was stable through seven freeze-thaw cycles. [Table 13]

[0229] 3.9 Donnan effect The Donnan effect (unequal distribution on both sides of a semipermeable membrane in the presence of impermeable charged ions) was assessed by measuring the pH of in-process samples taken from each diavolume during diafiltration (DF). The pH of the DF buffer, conditioning buffer, recovery pool, dilution pool, and conditioning material was also measured. UPLC analysis for the free histidine assay was used to measure the L-histidine concentration in the LSPD of selected samples.

[0230] The results of this experiment are summarized in Figure 12. The Donnan effect was observed in the recovery pool at a protein concentration of approximately 50 g / L, resulting in a pH increase of approximately 0.2 (relative to the pH of 5.8 in DF buffer). After dilution and adjustment to a protein concentration of 10 g / L, a 5-fold dilution factor was used, returning the pH to the target pH of 5.8. The histidine concentration in the diluted and conditioned pools was approximately 10 mM.

[0231] Example 4: Ingredient selection based on formulation development The formulation development studies described in Example 3 above informed the selection of various ingredients (e.g., protein, surfactant, antioxidant, buffer, pH, sucrose, and container), as described below.

[0232] Protein The protein concentration was selected to accurately deliver the Phase III dose via the IV bag without the use of diluents. The fractional dose on Day 1 was expected to be as low as 0.8-1.0 mg. The IV bag shaking study in Example 3.1 demonstrated that a low DP protein concentration was necessary to ensure sufficient surfactant was present in the diluted dose solution to protect the protein from mechanical agitation in order to deliver such a low dose. Furthermore, a lower protein concentration would allow for a larger dose volume and therefore more accurate dose delivery. For these reasons, the DP protein concentration was selected as 1 mg / ml. The DS concentration was selected as 10 mg / ml to balance a reasonable dilution factor from DS to DP with suitable facilities for DS storage.

[0233] 4.2.Surfactants Three surfactants were evaluated: PS20, super refined PS20 (srPS20), and P188. SrPS20 was excluded due to its high risk of inducing protein oxidation, fragmentation, and aggregation in low-protein concentration formulations (Example 3.2). The minimum required levels of PS20 and P188 were determined to be 0.05% (w / v) and 0.08% (w / v), respectively, from IV bag agitation studies (Example 3.1). Neither PS20 nor P188 showed any effect on protein quality under heat stress (Example 3.2). PS20 was selected as the surfactant type given its relatively low required concentration, the low protein concentration resulting in a low risk of ester bond hydrolysis (and therefore a low presence of any lipase impurities), and the low risk of surfactant oxidation due to the inclusion of an antioxidant. Based on the minimum required level determined from the IV bag shaking test (Example 3.1), a PS20 target concentration of 0.06% (w / v) was determined (a molar ratio of surfactant to mosunetuzumab of approximately 71 for a formulation containing 1 mg / ml of mosunetuzumab, and 0.05-0.07% (w / v)) as the DS release specification for PS20.

[0234] Antioxidants Oxidation was identified as a major risk for low protein concentration formulations of mosunetuzumab in knowledge-building studies, and L-Met protected the formulation from oxidation. During formulation development, oxidation was considered the primary risk evaluated, and a worst-case formulation for oxidation was challenged under heat stress and ambient light stress conditions (Example 3.3). These studies demonstrated that Met is required, but NAT provides no additional benefit with respect to light stress-induced oxidation.

[0235] The Met concentration was optimized in a hydrogen peroxide spike test using thermal stress (Example 3.4). In this stress model, 5 mM Met was required to fully protect the formulation from protein oxidation. Given that vaporized hydrogen peroxide is a common sterilization reagent for equipment such as isolators, DS and DP may experience challenges with residual hydrogen peroxide during the manufacturing process. Therefore, Met-mediated quenching of oxidation immediately after the addition of hydrogen peroxide is beneficial to the formulation's stability. To ensure the stability of DS and DP formulations against oxidative stress from multiple sources and to account for manufacturing variability in Met concentration, we selected a Met concentration of 10 mM in the DS and DP formulations.

[0236] 4.4.Buffer Histidine acetate (His) was used as a buffering species in Formulation 1 and demonstrated its ability to buffer DS and DP formulations at pH 5.5-6.1. Therefore, His was selected as the buffering species for the formulation. Knowledge-building tests showed that His concentrations in the 10-30 mM range positively affected acidic peak formation. Further testing demonstrated that formulations containing 10 mM His were less susceptible to oxidation compared to formulations containing 30 mM His in the absence of antioxidants (Example 3.3). His concentrations of 10 mM or less (e.g., 5 mM) were sufficient to buffer DS and DP solutions at pH 5.5-6.1. Long-term, real-time, accelerated, and stress-stable pH were confirmed in formulations containing 10 mM His (Examples 3.5 and 3.6). From a manufacturing perspective, given the pH shift observed in the Donnan effect evaluation (Example 3.8), His concentrations may shift in the recovery pool at 50 g / L. However, considering the high dilution factor from the recovered pool to the diluted pool (12.5 g / L) and DS (10 g / L), the His concentration returned to near 10 mM. Therefore, the impact of the Donnan effect on the His concentration in the final DS and DP is minimal. Taking all these factors into consideration, 10 mM histidine acetate was selected as the target buffer concentration.

[0237] 4.5.pH Long-term stability of early-stage formulations demonstrated that a target pH of 5.8 was appropriate for stability. Knowledge-building studies demonstrated that pH had a slight positive effect on acidic peak formation and no effect on aggregation or fragmentation. DP and DS screening studies on target and high-risk oxidation formulations demonstrated that pH (5.5-6.1) had minimal impact on real-time, accelerated, and stress stability (Examples 3.5 and 3.6). Based on these results, the formulation pH was selected as pH 5.8.

[0238] Sucrose Knowledge-building studies demonstrated that sucrose concentration did not show a significant effect on product quality attributes when high-risk oxidized formulations were excluded from statistical analysis. A relatively low concentration of sucrose (160 mM) was evaluated in DP and DS screening studies and in multiple freeze-thaw studies of the DS and was shown to have no effect on stability when compared to the target formulation (Examples 3.5, 3.6, 3.7).

[0239] 4.7. Container 316L stainless steel mini-cans were used for DS formulation screening. Multiple freeze-thaw tests showed adequate stability (Examples 3.6 and 3.7). Stainless steel is considered a high-risk container for protein degradation induced by metal leachates (e.g., compared to HASTELLOY®). These results suggest that the DS formulation is stable in stainless steel and HASTELLOY®. 316L stainless steel and HASTELLOY® mini-cans were evaluated in a DS representative test to provide additional supporting data to enable the use of either type of container for DS storage.

[0240] The DP vial configuration is required to support the delivery of a wide range of doses in clinical trials according to a dual-phase split-dosing scheme requiring two low fractional doses (e.g., 1 mg and 2 mg) and one full dose (e.g., 5-20 mg). To optimally support this dose range, a 20 ml vial with a nominal fill of 13.5 ml was selected.

[0241] IX. Embodiments Some embodiments of the technology described herein may be defined according to any of the following numbered embodiments.

[0242] 1. A pharmaceutical composition comprising mosunetuzumab, polysorbate 20 (PS20), methionine, a buffer, and a carrier, wherein the concentration of PS20 is 0.01% weight / volume to 0.1% weight / volume (w / v), the concentration of methionine is 1 mM to 50 mM, and the concentration of the buffer is 5 mM to 20 mM.

[0243] 2. The pharmaceutical composition of embodiment 1, wherein the concentration of mosunetuzumab is about 15 mg / ml or less.

[0244] 3. The pharmaceutical composition of embodiment 1 or 2, wherein the molar ratio of PS20 to mosunetuzumab is less than 100.

[0245] 4. The pharmaceutical composition of embodiment 3, wherein the molar ratio of PS20 to mosunetuzumab is 50-100.

[0246] 5. The pharmaceutical composition of embodiment 4, wherein the molar ratio of PS20 to mosunetuzumab is about 71.

[0247] 6. The pharmaceutical composition of any one of embodiments 1 to 5, wherein the concentration of mosunetuzumab is from about 0.5 mg / ml to about 2 mg / ml.

[0248] 7. The pharmaceutical composition of embodiment 6, wherein the concentration of mosunetuzumab is about 1 mg / ml.

[0249] 8. The pharmaceutical composition of any one of embodiments 1 to 7, which is formulated as a dosage form (DP).

[0250] 9. The pharmaceutical composition according to any one of embodiments 1 to 8, wherein the concentration of methionine is from about 2.5 mM to about 20 mM.

[0251] 10. The pharmaceutical composition of embodiment 9, wherein the concentration of methionine is about 10 mM.

[0252] 11. The pharmaceutical composition of any one of embodiments 1-10, wherein the buffering agent is histidine, phosphate, succinate, acetate, or a combination thereof.

[0253] 12. The pharmaceutical composition of embodiment 11, wherein the buffering agent is histidine.

[0254] 13. The pharmaceutical composition of embodiment 12, wherein the histidine is histidine acetate.

[0255] 14. The pharmaceutical composition according to any one of embodiments 1 to 13, wherein the concentration of the buffering agent is from about 8 mM to about 12 mM.

[0256] 15. The pharmaceutical composition of embodiment 14, wherein the concentration of the buffering agent is about 10 mM.

[0257] 16. The pharmaceutical composition of any one of embodiments 1-15, wherein the buffering agent is histidine acetate at a concentration of about 8 mM to about 12 mM.

[0258] 17. The pharmaceutical composition of embodiment 16, wherein the concentration of histidine acetate is about 10 mM.

[0259] 18. The pharmaceutical composition according to any one of embodiments 1 to 17, further comprising an isotonicity agent.

[0260] 19. The pharmaceutical composition according to embodiment 18, wherein the isotonicity agent is a sugar, an amino acid or a salt.

[0261] 20. The pharmaceutical composition according to embodiment 19, wherein the tonicity agent is a sugar.

[0262] 21. The pharmaceutical composition of embodiment 20, wherein the sugar is sucrose, glucose, glycerol or trehalose.

[0263] 22. The pharmaceutical composition according to embodiment 21, wherein the sugar is sucrose.

[0264] 23. The pharmaceutical composition according to any one of embodiments 18 to 22, wherein the tonicity agent is at a concentration of about 100 mM to about 500 mM.

[0265] 24. The pharmaceutical composition of embodiment 23, wherein the concentration of the tonicity agent is from about 200 mM to about 300 mM.

[0266] 25. The pharmaceutical composition of embodiment 24, wherein the concentration of the tonicity agent is about 240 mM.

[0267] 26. The pharmaceutical composition of any one of embodiments 1 to 25, having a pH of about 4.5 to about 8.

[0268] 27. The pharmaceutical composition of embodiment 26, having a pH of about 5.5 to about 6.1.

[0269] 28. The pharmaceutical composition of embodiment 27, wherein the pH is about 5.8.

[0270] 29. The pharmaceutical composition of any one of embodiments 1-28, wherein mosunetuzumab has a methionine at position 257 (EU numbering) of the Fc region, and wherein oxidation of the methionine at position 257 of the Fc region is less than about 10% over 2 weeks at 40°C.

[0271] 30. The pharmaceutical composition of embodiment 29, wherein the oxidation of methionine at position 257 of the Fc region is less than or equal to about 6% over a period of 2 weeks at 40°C.

[0272] 31. A pharmaceutical composition comprising mosunetuzumab, a surfactant, methionine, and a carrier, wherein the composition has a pH of about 5.8; (i) the concentration of mosunetuzumab is about 10 mg / ml or less; (ii) the concentration of the surfactant is about 0.05% to about 0.1% (w / v); (iii) A pharmaceutical composition, wherein the concentration of methionine is about 10 mM.

[0273] 32. The pharmaceutical composition according to embodiment 31, wherein the molar ratio of surfactant to mosunetuzumab is 100 or less.

[0274] 33. The pharmaceutical composition according to embodiment 31 or 32, wherein the surfactant is PS20 or poloxamer 188 (P188).

[0275] 34. The pharmaceutical composition according to embodiment 33, wherein the surfactant is PS20 and the concentration of PS20 is about 0.06% (w / v).

[0276] 35. The pharmaceutical composition of embodiment 34, wherein the molar ratio of PS20 to mosunetuzumab is about 50 to about 100.

[0277] 36. The pharmaceutical composition of embodiment 35, wherein the molar ratio of PS20 to mosunetuzumab is about 71.

[0278] 37. The pharmaceutical composition according to embodiment 33, wherein the surfactant is P188 and the concentration of P188 is about 0.1% (w / v).

[0279] 38. The pharmaceutical composition of embodiment 37, wherein the molar ratio of P188 to mosunetuzumab is from about 5 to about 25.

[0280] 39. The pharmaceutical composition according to embodiment 38, wherein the molar ratio of P188 to mosunetuzumab is about 17.

[0281] 40. The pharmaceutical composition of any one of embodiments 31-39, wherein the concentration of mosunetuzumab is from about 0.5 mg / ml to about 2 mg / ml.

[0282] 41. The pharmaceutical composition of embodiment 40, wherein the concentration of mosunetuzumab is about 1 mg / ml.

[0283] 42. A pharmaceutical composition according to any one of embodiments 35 to 41, formulated as a DP.

[0284] 43. The pharmaceutical composition of any one of embodiments 31-42, further comprising histidine acetate at a concentration of about 10 mM and / or sucrose at a concentration of about 240 mM.

[0285] 44. The pharmaceutical composition of any one of embodiments 1 to 43, which is in unit dosage form.

[0286] 45. The pharmaceutical composition according to embodiment 44, wherein the unit dosage form is a liquid formulation for dilution.

[0287] 46. ​​The pharmaceutical composition according to embodiment 45, wherein the liquid formulation for dilution is supplied in a container having a volume of about 50 ml.

[0288] 47. The pharmaceutical composition according to embodiment 45, wherein the liquid formulation for dilution is supplied in a container having a volume of about 2 ml.

[0289] 48. The pharmaceutical composition according to embodiment 45 or 46, wherein the volume of the liquid formulation for dilution is 20 to 40 ml.

[0290] 49. The pharmaceutical composition according to embodiment 48, wherein the volume of the liquid formulation for dilution is about 30 ml.

[0291] 50. The pharmaceutical composition according to embodiment 45 or 47, wherein the volume of the liquid formulation for dilution is 0.2 to 2 ml.

[0292] 51. The pharmaceutical composition according to embodiment 50, wherein the volume of the liquid formulation for dilution is about 1 ml.

[0293] 52. The pharmaceutical composition according to any one of embodiments 45 to 51, wherein the liquid formulation is for dilution with saline containing 0.45% or 0.9% (w / v) NaCl.

[0294] 53. A pharmaceutical composition according to any one of embodiments 1-52, comprising no more than 1,000 particles per ml having a diameter of 2 μm or greater as detected by high accuracy liquid particle counting (HIAC).

[0295] 54. The pharmaceutical composition according to any one of embodiments 1-53, wherein the carrier is water.

[0296] 55. The pharmaceutical composition of any one of embodiments 1-54, having a shelf life of at least 36 months when stored at 55.5°C ± 3°C and protected from light.

[0297] 56. The pharmaceutical composition of any one of embodiments 1-55, which is stable through one or more freeze-thaw cycles.

[0298] 57. The pharmaceutical composition of embodiment 56, which is stable through 3 or more freeze-thaw cycles.

[0299] 58. The pharmaceutical composition of any one of embodiments 1-57, which is stable at about 25°C for about 2 weeks or more.

[0300] 59. The pharmaceutical composition of embodiment 58, which is stable at about 25°C for about 4 weeks or more.

[0301] 60. The pharmaceutical composition of any one of embodiments 1 to 59, which is stable at −20° C. for about 48 months or more.

[0302] 61. The pharmaceutical composition according to any one of embodiments 56-60, wherein stability is assessed by size-exclusion high performance liquid chromatography (SE-HPLC).

[0303] 62. The pharmaceutical composition of embodiment 61, which is determined to be stable if it maintains a purity that varies less than 5% as measured by SE-HPLC.

[0304] 63. The pharmaceutical composition according to any one of embodiments 56-60, wherein stability is assessed by a non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay.

[0305] 64. The pharmaceutical composition of embodiment 63, which is determined to be stable if it maintains a purity that changes less than 5% as measured by a non-reducing CE-SDS assay.

[0306] 65. The pharmaceutical composition according to embodiment 63 or 64, wherein the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

[0307] 66. A pharmaceutical composition according to any one of embodiments 1 to 65, having a purity of about 85% or greater as assessed by SE-HPLC.

[0308] 67. The pharmaceutical composition of embodiment 66, having a purity of about 90% or greater as assessed by SE-HPLC.

[0309] 68. The pharmaceutical composition of embodiment 67, having a purity of about 95% or greater as assessed by SE-HPLC.

[0310] 69. A pharmaceutical composition according to any one of embodiments 66-68, wherein the purity as assessed by SE-HPLC remains approximately the same for about 36 months or more at about 5°C.

[0311] 70. The pharmaceutical composition of embodiment 69, wherein the purity as assessed by SE-HPLC remains approximately the same for about 42 months or more at about 5°C.

[0312] 71. The pharmaceutical composition of embodiment 70, wherein the purity as assessed by SE-HPLC remains approximately the same for about 64 months or more at about 5°C.

[0313] 72. The pharmaceutical composition of any one of embodiments 1-71, having a purity of about 75% or greater as assessed by a non-reducing CE-SDS assay.

[0314] 73. The pharmaceutical composition of embodiment 72, having a purity of about 80% or greater as assessed by a non-reducing CE-SDS assay.

[0315] 74. The pharmaceutical composition of embodiment 73, having a purity of about 85% or greater as assessed by a non-reducing CE-SDS assay.

[0316] 75. The pharmaceutical composition of any one of embodiments 72-74, wherein purity as assessed by a non-reducing CE-SDS assay is maintained for about 36 months or more at about 5°C.

[0317] 76. The pharmaceutical composition of embodiment 75, wherein the purity as assessed by a non-reducing CE-SDS assay is maintained for about 42 months or more at about 5°C.

[0318] 77. The pharmaceutical composition according to any one of embodiments 72-76, wherein the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

[0319] 78. The pharmaceutical composition of any one of embodiments 1-77, formulated for intravenous administration.

[0320] 79. The pharmaceutical composition of any one of embodiments 1-78, which does not contain a preservative.

[0321] 80.1 mg / ml mosunetuzumab, 10 mM L-histidine acetate, 240 mM sucrose, 0.06% (w / v) PS20, and 10 mM methionine, pH 5.8, formulated for administration by infusion after dilution with saline containing 0.45% or 0.9% NaCl.

[0322] 81. A pharmaceutical composition according to any one of embodiments 1 to 80, for use as a medicament.

[0323] 82. A pharmaceutical composition according to any one of embodiments 1 to 80, for use in treating or delaying the progression of cancer in a subject in need thereof.

[0324] 83. A pharmaceutical composition according to any one of embodiments 1 to 80, for use in enhancing immune function in a subject with cancer.

[0325] 84. The pharmaceutical composition according to any one of embodiments 1 to 80 for use in treating or delaying the progression of cancer or for use in enhancing immune function in a subject with cancer, wherein the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma. B-cell lymphoma with leukemia, germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma B-cell lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, B-cell leukemia, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal lymphoma 1. The pharmaceutical composition of claim 1, wherein the non-Hodgkin's lymphoma is selected from the group consisting of: lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, and primary effusion lymphoma.

[0326] 85. The pharmaceutical composition for use according to embodiment 84, wherein the NHL is GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL or BL.

[0327] 86. The pharmaceutical composition for use according to embodiment 85, wherein the NHL is FL.

[0328] 87. The pharmaceutical composition for use according to embodiment 86, wherein the FL is relapsed and / or refractory (R / R).

[0329] 88. The pharmaceutical composition for use according to embodiment 87, wherein the subject with R / R FL has relapsed after or is refractory to at least two prior systemic therapies.

[0330] 89. The pharmaceutical composition for use according to embodiment 88, wherein the subject has received prior systemic therapy comprising an anti-CD20 monoclonal antibody.

[0331] 90. The pharmaceutical composition for use according to embodiment 88 or 89, wherein the subject has received prior systemic therapy comprising an alkylating agent.

[0332] 91. The pharmaceutical composition for use according to any one of embodiments 82-90, wherein mosunetuzumab is formulated for administration to a subject at a dose of from about 0.1 mg to about 100 mg.

[0333] 92. The pharmaceutical composition for use according to embodiment 91, wherein mosunetuzumab is formulated for administration to a subject at a dose of from about 1 mg to about 60 mg.

[0334] 93. The pharmaceutical composition for use according to embodiment 92, wherein mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg, 2 mg, 6 mg, 9 mg, 13.5 mg, 20 mg, 30 mg or 60 mg.

[0335] 94. The pharmaceutical composition for use according to embodiment 93, wherein mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg, 2 mg, 30 mg or 60 mg.

[0336] 95. The pharmaceutical composition for use according to any one of embodiments 82 to 94, which is formulated for administration to a subject after dilution with saline containing 0.45% or 0.9% (w / v) NaCl.

[0337] 96. The pharmaceutical composition for use according to embodiment 95, wherein the concentration of mosunetuzumab after dilution with saline is from about 0.01 mg / ml to about 0.3 mg / ml.

[0338] 97. The pharmaceutical composition for use according to embodiment 96, wherein after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0339] 98. The pharmaceutical composition for use according to any one of embodiments 82-97, which is for use in combination with at least one additional therapeutic agent.

[0340] 99. The pharmaceutical composition for use according to embodiment 98, wherein the at least one additional therapeutic agent comprises a PD-1 axis binding antagonist.

[0341] 100. The pharmaceutical composition for use according to embodiment 99, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

[0342] 101. The pharmaceutical composition for use according to embodiment 100, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0343] 102. The pharmaceutical composition for use according to embodiment 101, wherein the PD-L1 binding antagonist is selected from the group consisting of atezolizumab (MPDL3280A), MDX-1105 (BMS-936559) and MEDI4736 (durvalumab).

[0344] 103. The pharmaceutical composition for use according to embodiment 100, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0345] 104. The pharmaceutical composition for use according to embodiment 103, wherein the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), AMG404, REGN2810 (cemiplimab) and AMP-224.

[0346] 105. The pharmaceutical composition for use according to embodiment 100, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0347] 106. The pharmaceutical composition for use according to embodiment 105, wherein the PD-L2 binding antagonist is an antibody or immunoadhesin.

[0348] 107. The pharmaceutical composition for use according to embodiment 98, wherein the at least one additional therapeutic agent comprises obinutuzumab, rituximab, a corticosteroid or tocilizumab.

[0349] 108. The pharmaceutical composition for use according to embodiment 98, wherein the at least one additional therapeutic agent comprises an antibody-drug conjugate (ADC).

[0350] 109. The pharmaceutical composition for use according to embodiment 108, wherein the ADC is an anti-CD79b ADC.

[0351] 110. The pharmaceutical composition for use according to embodiment 109, wherein the anti-CD79b ADC is polatuzumab vedotin.

[0352] 111. The pharmaceutical composition for use according to any one of embodiments 82 to 110, which is formulated for intravenous administration.

[0353] 112. The pharmaceutical composition for use according to any one of embodiments 82 to 111, wherein the subject is a human.

[0354] 113. A method for treating or delaying the progression of cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments 1-80.

[0355] 114. A method for enhancing immune function in a subject with cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments 1-80.

[0356] 115. A method for treating or delaying the progression of cancer in a subject in need thereof, or for enhancing immune function in a subject with cancer, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments 1 to 80, wherein the cancer is selected from the group consisting of CLL, B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, GCB DLBCL, ABC DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, B-cell leukemia, FL, MCL, AML, MZL, SLL, The method of claim 1, wherein the NHL is selected from the group consisting of LL, WM, CNSL, BL, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, MALT lymphoma, nodal marginal zone lymphoma, pediatric nodular marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma, and primary effusion lymphoma.

[0357] 116. The method of embodiment 115, wherein the NHL is GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL or BL.

[0358] 117. The method of embodiment 116, wherein the NHL is FL.

[0359] 118. The method of embodiment 117, wherein the FL is relapsed and / or refractory (R / R).

[0360] 119. The method of embodiment 118, wherein the subject with R / R FL has relapsed after or is refractory to at least two prior systemic therapies.

[0361] 120. The method of embodiment 119, wherein the subject has received prior systemic therapy including an anti-CD20 monoclonal antibody.

[0362] 121. The method of embodiment 119 or 120, wherein the subject has received prior systemic therapy including an alkylating agent.

[0363] 122. The method of any one of embodiments 113-121, wherein mosunetuzumab is administered to the subject at a dose of about 0.1 mg to about 100 mg.

[0364] 123. The method of embodiment 122, wherein mosunetuzumab is administered at a dose of about 1 mg to about 60 mg.

[0365] 124. The method of embodiment 123, wherein mosunetuzumab is administered to the subject at a dose of about 1 mg, 2 mg, 6 mg, 9 mg, 13.5 mg, 20 mg, 30 mg, or 60 mg.

[0366] 125. The method of embodiment 124, wherein mosunetuzumab is administered to the subject at a dose of about 1 mg, 2 mg, 30 mg, or 60 mg.

[0367] 126. The method according to any one of embodiments 113 to 125, wherein the pharmaceutical composition is administered to the subject after dilution with saline containing 0.45% or 0.9% (w / v) NaCl.

[0368] 127. The method of embodiment 126, wherein the concentration of mosunetuzumab after dilution with saline is from about 0.01 mg / ml to about 0.3 mg / ml.

[0369] 128. The method of embodiment 127, wherein after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0370] 129. The method of any one of embodiments 113-128, wherein mosunetuzumab is administered to the subject in a dosing regimen comprising at least three 21-day dosing cycles; (a) a first 21-day dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of mosunetuzumab administered to the subject on days 1, 8, and 15, respectively, of the first dosing cycle, wherein C1D1 is about 1 mg, C1D2 is about 2 mg, and C1D3 is about 60 mg; (b) the second dosing cycle comprises a single dose (C2D1) of mosunetuzumab administered to the subject on day 1 of the second dosing cycle, wherein C2D1 is about 60 mg; (c) The method, wherein the third dosing cycle comprises a single dose (C3D1) of mosunetuzumab administered to the subject on day 1 of the third dosing cycle, wherein C3D1 is about 30 mg.

[0371] 130. The method of embodiment 129, wherein the dosing regimen comprises 1 to 14 additional dosing cycles, each comprising an additional single dose of about 30 mg of mosunetuzumab.

[0372] 131. The method of embodiment 130, wherein the administration regimen comprises 1 to 5 additional administration cycles.

[0373] 132. The method of embodiment 131, wherein the administration regimen comprises five additional administration cycles.

[0374] 133. The method of any one of embodiments 130-132, wherein each additional single dose of mosunetuzumab is administered to the subject on day 1 of each additional administration cycle.

[0375] 134. The method of any one of embodiments 113-133, wherein the subject is co-administered with at least one additional therapeutic agent.

[0376] 135. The method of embodiment 134, wherein the at least one additional therapeutic agent comprises a PD-1 axis binding antagonist.

[0377] 136. The method of embodiment 135, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

[0378] 137. The method of embodiment 136, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0379] 138. The method of embodiment 137, wherein the PD-L1 binding antagonist is selected from the group consisting of atezolizumab (MPDL3280A), MDX-1105 (BMS-936559), and MEDI4736 (durvalumab).

[0380] 139. The use according to embodiment 136, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0381] 140. The method of embodiment 139, wherein the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), AMG404, REGN2810 (cemiplimab), and AMP-224.

[0382] 141. The method of embodiment 136, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0383] 142. The method of embodiment 141, wherein the PD-L2 binding antagonist is an antibody or immunoadhesin.

[0384] 143. The method of embodiment 134, wherein the at least one additional therapeutic agent comprises obinutuzumab, rituximab, a corticosteroid, or tocilizumab.

[0385] 144. The method of embodiment 134, wherein at least one additional therapeutic agent comprises an ADC.

[0386] 145. The method of embodiment 144, wherein the ADC is an anti-CD79b ADC.

[0387] 146. The method of embodiment 145, wherein the anti-CD79b ADC is polatuzumab vedotin.

[0388] 147. The method according to any one of embodiments 113-146, wherein the pharmaceutical composition is administered intravenously.

[0389] 148. The method of any one of embodiments 113-147, wherein the subject is a human.

[0390] 149. Use of a pharmaceutical composition according to any one of embodiments 1 to 80 in the manufacture of a medicament for treating or delaying the progression of cancer in a subject in need thereof.

[0391] 150. Use of a pharmaceutical composition according to any one of embodiments 1 to 80 in the manufacture of a medicament for enhancing immune function in a subject with cancer.

[0392] 151. Use of the pharmaceutical composition according to any one of embodiments 1 to 80 for treating or delaying the progression of cancer in a subject in need thereof.

[0393] 152. Use of a pharmaceutical composition according to any one of embodiments 1 to 80 for enhancing immune function in a subject with cancer.

[0394] 153. The cancer is CLL, B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, GCB DLBCL, ABC DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, B-cell leukemia, FL, MCL, AML, MZL, SLL, LL, WM, CNSL, Use according to any one of embodiments 149 to 152, wherein the NHL is selected from the group consisting of BL, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, hairy cell leukemia variant, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, MALT lymphoma, nodular marginal zone lymphoma, childhood nodular marginal zone lymphoma, childhood follicular lymphoma, lymphomatoid granulomatosis, plasmablastic lymphoma and primary effusion lymphoma.

[0395] 154. The use according to embodiment 153, wherein the NHL is GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL or BL.

[0396] 155. The use according to embodiment 154, wherein the NHL is FL.

[0397] 156. The use according to embodiment 155, wherein the FL is relapsed and / or refractory (R / R).

[0398] 157. The use according to embodiment 156, wherein the subject with R / R FL has relapsed after or is refractory to at least two prior systemic therapies.

[0399] 158. The use according to embodiment 157, in which the patient has received prior systemic therapy including an anti-CD20 monoclonal antibody.

[0400] 159. The method of embodiment 157 or 158, wherein the subject has received prior systemic therapy including an alkylating agent.

[0401] 160. The use according to any one of embodiments 149-159, wherein mosunetuzumab is formulated for administration to a subject at a dose of from about 0.1 mg to about 100 mg.

[0402] 161. The use of embodiment 160, wherein mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg to about 60 mg.

[0403] 162. The use of embodiment 161, wherein mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg, 2 mg, 6 mg, 9 mg, 13.5 mg, 20 mg, 30 mg, or 60 mg.

[0404] 163. The use of embodiment 162, wherein mosunetuzumab is formulated for administration to a subject at a dose of about 1 mg, 2 mg, 30 mg, or 60 mg.

[0405] 164. The use according to any one of embodiments 149 to 163, wherein the pharmaceutical composition is formulated for administration to a subject after dilution with saline containing 0.45% or 0.9% (w / v) NaCl.

[0406] 165. The use according to embodiment 164, wherein the concentration of mosunetuzumab after dilution with saline is from about 0.01 mg / ml to about 0.3 mg / ml.

[0407] 166. The use according to embodiment 165, wherein after dilution with saline, the concentration of mosunetuzumab is about 0.01 mg / ml, about 0.02 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml.

[0408] 167. The use according to any one of embodiments 149-166, wherein the subject is co-administered with at least one additional therapeutic agent.

[0409] 168. The use according to embodiment 167, wherein the at least one additional therapeutic agent comprises a PD-1 axis binding antagonist.

[0410] 169. The use according to embodiment 168, wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.

[0411] 170. The use according to embodiment 169, wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0412] 171. The use of embodiment 170, wherein the PD-L1 binding antagonist is selected from the group consisting of atezolizumab (MPDL3280A), MDX-1105 (BMS-936559) and MEDI4736 (durvalumab).

[0413] 172. The use according to embodiment 169, wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0414] 173. The use according to embodiment 172, wherein the PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (lambrolizumab), AMG404, REGN2810 (cemiplimab) and AMP-224.

[0415] 174. The use according to embodiment 169, wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0416] 175. The use of embodiment 174, wherein the PD-L2 binding antagonist is an antibody or immunoadhesin.

[0417] 176. The use according to embodiment 167, wherein the at least one additional therapeutic agent comprises obinutuzumab, rituximab, corticosteroids or tocilizumab.

[0418] 177. The use of embodiment 167, wherein at least one additional therapeutic agent comprises an ADC.

[0419] 178. The use according to embodiment 177, wherein the ADC is an anti-CD79b ADC.

[0420] 179. The use according to embodiment 178, wherein the anti-CD79b ADC is polatuzumab vedotin.

[0421] 180. The use according to any one of embodiments 149 to 179, wherein the pharmaceutical composition is administered intravenously.

[0422] 181. The use according to any one of embodiments 149-180, wherein the subject is a human.

[0423] 182. A pharmaceutical composition for use according to any one of embodiments 82 to 97, or a use according to any one of embodiments 149 to 166, wherein mosunetuzumab is administered to the subject in a dosing regimen comprising at least three 21-day dosing cycles, (a) a first 21-day dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of mosunetuzumab administered to the subject on days 1, 8, and 15, respectively, of the first dosing cycle, wherein C1D1 is about 1 mg, C1D2 is about 2 mg, and C1D3 is about 60 mg; (b) the second dosing cycle comprises a single dose (C2D1) of mosunetuzumab administered to the subject on day 1 of the second dosing cycle, wherein C2D1 is about 60 mg; (c) The pharmaceutical composition for use, or the use, wherein the third administration cycle comprises a single dose (C3D1) of mosunetuzumab administered to the subject on day 1 of the third administration cycle, wherein C3D1 is about 30 mg.

[0424] 183. The pharmaceutical composition for use or use according to embodiment 182, wherein the dosing regimen comprises 1 to 14 additional dosing cycles, each comprising an additional single dose of mosunetuzumab of about 30 mg.

[0425] 184. The pharmaceutical composition for use or use according to embodiment 183, wherein the administration regimen comprises 1 to 5 additional administration cycles.

[0426] 185. The pharmaceutical composition for use or use according to embodiment 184, wherein the administration regimen comprises five additional administration cycles.

[0427] 186. The pharmaceutical composition for use or use according to any one of embodiments 182-185, wherein each additional single dose of mosunetuzumab is administered to the subject on day 1 of each additional administration cycle.

[0428] 187. The pharmaceutical composition for use or use according to any one of embodiments 182 to 186, wherein the pharmaceutical composition is administered intravenously.

[0429] 188. The pharmaceutical composition for use or the use according to any one of embodiments 182 to 187, wherein the subject is a human.

[0430] Other embodiments The foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition comprising approximately 1 mg / mL mosunetuzumab, approximately 10 mM L-histidine, approximately 240 mM sucrose, approximately 0.06% (w / v) PS20, and approximately 10 mM methionine, wherein the pharmaceutical composition has a pH of 5.5 to 6.

1.

2. The pharmaceutical composition described in claim 1, wherein the pharmaceutical composition is in unit dosage form.

3. The pharmaceutical composition of claim 1, wherein mosunetuzumab has methionine at position 257 (EU numbering) of the Fc region, and oxidation of the methionine at position 257 of the Fc region is less than approximately 10% at 40°C over a period of two weeks.

4. The pharmaceutical composition of claim 2, wherein the unit dosage form is a liquid formulation for dilution.

5. The pharmaceutical composition of claim 4, wherein the liquid formulation for dilution is supplied in a container having a volume of approximately 50 ml.

6. A pharmaceutical composition comprising: (a) has a shelf life of at least 36 months when stored at 5°C ± 3°C and protected from light; (b) stable through one or more freeze-thaw cycles; (c) stable for at least about 2 weeks at about 25°C; or (d) stable for about 48 months or more at −20° C.; The pharmaceutical composition of claim 1.

7. The pharmaceutical composition of claim 6, wherein the stability is assessed by size-exclusion high performance liquid chromatography (SE-HPLC) or by a non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay. (a) A pharmaceutical composition is determined to be stable if it maintains a purity that changes less than 5% as measured by SE-HPLC. (b) the pharmaceutical composition has a purity of about 85% or greater as assessed by SE-HPLC; (c) the purity of the pharmaceutical composition, as assessed by SE-HPLC, remains substantially the same for greater than about 36 months at about 5°C; (d) a pharmaceutical composition is determined to be stable if it maintains a purity that changes less than 5% as measured by a non-reducing CE-SDS assay; (e) the pharmaceutical composition has a purity of about 75% or greater as assessed by a non-reducing CE-SDS assay; or (f) the purity of the pharmaceutical composition, as assessed by a non-reducing CE-SDS assay, is maintained for about 36 months or more at about 5°C; The pharmaceutical composition of claim 7.

9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for administration by injection after dilution with normal saline containing 0.45% or 0.9% NaCl.

10. The pharmaceutical composition of claim 1, further comprising histidine acetate.

11. The pharmaceutical composition described in claim 1, wherein the pharmaceutical composition is formulated as a drug product (DP).

12. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for intravenous administration.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is formulated for administration by injection.

14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition does not contain a preservative.

15. The pharmaceutical composition described in claim 4, wherein the volume of the liquid preparation for dilution is 20 to 40 ml.

16. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition contains 1,000 or less particles having a diameter of 2 μm or more per ml as detected by high accuracy particle counting (HIAC).

17. The pharmaceutical composition described in claim 4, wherein the volume of the liquid preparation for dilution is 0.2 to 2 ml.

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