Pharmaceutical compositions and methods of use of therapeutic proteins

Abstract

The present disclosure provides pharmaceutical compositions of therapeutic proteins and methods of using same.

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. The XML copy, created on March 22, 2023, is named 50474-276WO2_Sequence_Listing_3_22_23 and is 119,559 bytes in size.

[0002] FIELD OF THE INVENTION The present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising therapeutic proteins (e.g., T cell-dependent bispecific antibodies or T cell-engaging bispecific antibodies) and methods of use thereof. [Background technology]

[0003] background Therapeutic proteins, such as antibodies (e.g., bispecific antibodies; e.g., T cell-dependent bispecific antibodies (TDBs) or T cell-engaging bispecific antibodies (TCBs); e.g., anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 TDBs or TCBs; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab), are increasingly being used to treat various diseases and disorders, such as cancer and autoimmune disorders. Such therapeutic proteins can be formulated in aqueous carriers for administration to subjects, for example, by intravenous or subcutaneous administration. During storage, handling, and administration of such pharmaceutical compositions, it is necessary to mitigate potential losses of therapeutic proteins due to degradation and surface adsorption, such as adsorption of proteins to the surfaces of filters, storage canisters, tubing, syringes, intravenous fluid bags, and other containers. When a pharmaceutical composition contains a relatively low concentration of therapeutic protein, protein loss can be dramatically increased by these factors, resulting in a reduced therapeutic effect of the pharmaceutical composition.

[0004] Thus, there is a need in the art to develop pharmaceutical formulations in which therapeutic proteins (eg, low dose therapeutic proteins) are stable and protected from loss due to adsorption. Summary of the Invention

[0005] Disclosure Overview The present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising a therapeutic protein (e.g., a therapeutic protein at low concentrations, such as a bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., an anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody (TDB) or a T cell-engaging bispecific antibody (TCB); e.g., an anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab)), and methods of use thereof. The disclosed compositions and related methods address the problem of delivering therapeutic proteins formulated at low concentrations, ensuring that patients receive the intended dose of therapeutic protein with little or no protein loss during storage and administration.

[0006] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody (TDB) or a T cell-engaging bispecific antibody (TCB); e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab), a surfactant (e.g., polysorbate 20 (PS20) or poloxamer 188 (P188)), methionine, a buffering agent, and a carrier. In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to therapeutic protein 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, 50 to 100, 60 to 80, 70 to 80, or 40 to 50. In certain embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to therapeutic protein is 1 to 100. In some embodiments, the molar ratio of detergent (e.g., PS20) to therapeutic protein is 45-100, 45-55, 50-100, 60-90, 70-90, 60-80, 70-80, 65-75, or 75-85, e.g., about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In a particular embodiment, the molar ratio of detergent to therapeutic protein is about 71. In another particular embodiment, the molar ratio of detergent to therapeutic protein is about 79. In yet another particular embodiment, the molar ratio of detergent to therapeutic protein is about 48. In other embodiments, the molar ratio of surfactant (e.g., P188) to therapeutic protein is 5-50, 5-25, 10-15, or 15-20, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the molar ratio of surfactant (e.g., P188) to therapeutic protein is about 14.In another specific embodiment, the molar ratio of surfactant (eg, P188) to therapeutic protein is about 11.5.

[0007] In some embodiments, the concentration of surfactant (e.g., PS20 or P188) is between 0.01% and 0.12% weight / volume (e.g., 0.01% to 0.025%, 0.025% to 0.5%, 0.05% to 0.075%, or 0.075% to 0.12% (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 0.12%, or 0.09% to 0.12% (w / v). 0.08% to 0.09%, or 0.09% to 0.12% (w / v); for example, 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%, about 0.115%, or about 0.12% (w / v).

[0008] 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, , 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 methionine is about 10 mM.

[0009] 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, or 9 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).

[0010] In some embodiments, the concentration of a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody or a T cell-engaging bispecific antibody; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is 10 mg / ml or less (e.g., 9 mg / ml or less, 8 mg / ml or less, 7.5 mg / ml or less, 7 mg / ml or less, 6 mg / ml or less, 5 mg / ml or less, 4 mg / ml or less, 3 mg / ml or less, 2.5 mg / ml or less, 2 mg / ml or less, 1.5 mg / ml or less, 1.0 mg / ml or less, 0.5 mg / ml or less, 0.25 mg / ml or less, 0.2 mg / ml or less, or less, or 0.1 mg / ml or less, e.g., 0.01 mg / ml to 0.1 mg / ml, 0.1 mg / ml to 1 mg / ml, 1 mg / ml to 5 mg / ml, or 5 mg / ml to 10 mg / ml, e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, 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 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml. In certain embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In another specific embodiment, the concentration of the therapeutic protein is about 3 mg / ml.

[0011] In some embodiments, the pharmaceutical composition is formulated as a drug product (DP). In some embodiments, the pharmaceutical composition formulated as a DP has a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody or a T cell-engaging bispecific antibody; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) concentration of about 1 mg / ml, and / or 45-100, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140, 140-160, 160-200, 180-220, 180-240, 180-260, 180-300, 180-320, 180-400, 180-420, 180-520, 180-540, 180-640, 180-700, 180-820, 180-900, 180-190, 190-210, 190-220, 190-230, 190-340, 190-350, 190-410, 190-420, 190-540, 190-650, 190-700, 190-820, 190-900, 190-190, 190-240, 190-250, 190-350, 190-420, 190-540, The molar ratio of surfactant (e.g., PS20) to therapeutic protein is 5, 50-100, 60-90, 70-90, 60-80, 70-80, 65-75, or 75-85, e.g., about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In certain embodiments, the molar ratio of surfactant to therapeutic protein (e.g., mosunetuzumab or lunimotamab) is about 71. In another specific embodiment, the molar ratio of surfactant to therapeutic protein (e.g., glofitamab) is about 79. In some embodiments, the molar ratio of surfactant to therapeutic protein (e.g., lunimotamab) is about 48.

[0012] In some embodiments, the buffering agent is histidine, phosphate, succinate, acetate, or a combination thereof. For example, in some embodiments, the buffering agent is histidine, such as histidine acetate or histidine hydrochloride (HCl). The concentration of the buffering agent (e.g., histidine, e.g., histidine acetate or histidine HCl, e.g., L-histidine acetate or L-histidine HCl) 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 some embodiments, the concentration of the buffering agent (e.g., histidine, e.g., histidine acetate or histidine HCl, e.g., L-histidine acetate or L-histidine HCl) is about 10 mM or 20 mM. In certain embodiments, the buffering agent is histidine HCl at a concentration of about 20 mM. In another specific embodiment, the buffering agent is histidine acetate at a concentration of about 20 mM. In yet another specific embodiment, the buffering agent is histidine acetate at a concentration of about 10 mM.

[0013] In some embodiments, the pharmaceutical composition further comprises a tonicity agent such as a sugar, an amino acid, or a salt. In embodiments in which the tonicity agent is a sugar, the sugar may be, for example, sucrose, glucose, glycerol, or trehalose. In certain embodiments, the sugar is sucrose. In some embodiments, the tonicity agent (e.g., a sugar, such as sucrose) is present in a concentration of 100 mM to 500 mM (e.g., 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 500 mM, 500 mM to 600 mM, 600 mM to 700 mM, 700 mM to 800 mM, 800 mM to 900 mM, 900 mM to 1000 mM, 900 mM to 1100 mM, 900 mM to 1200 mM, 900 mM to 1300 mM, 900 mM to 1400 mM, 900 mM to 1500 mM, 900 mM to 1600 mM, 900 mM to 1800 mM, 900 mM to 19 ... The concentration is, for example, 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 tonicity agent (eg, a sugar, such as sucrose) is about 240 mM.

[0014] In some embodiments, the pharmaceutical composition further comprises an antioxidant. In some embodiments, the antioxidant is N-acetyl-DL-tryptophan. In some embodiments, the concentration of N-acetyl-DL-tryptophan is 0.1 mM to 0.5 mM (e.g., about 0.1 mM, about 0.15 mM, about 0.2 mM, about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.45 mM, or about 0.5 mM). In some embodiments, the concentration of N-acetyl-DL-tryptophan is about 0.3 mM.

[0015] 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 5.1 to 6.1. In certain embodiments, the pharmaceutical composition has a pH of about 5.8. In another particular embodiment, the pharmaceutical composition has a pH of about 5.5.

[0016] In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is an anti-CD3 antibody. In some embodiments, the therapeutic protein is a bispecific antibody. For example, the bispecific antibody can be a T cell-dependent bispecific antibody (TDB) or a T cell-engaging bispecific antibody (TCB), e.g., a bispecific antibody having at least one target antigen-binding portion and one CD3-binding portion. In some embodiments, the target antigen-binding portion of the bispecific antibody (e.g., TDB or TCB) can bind to CD20, FcRH5, or HER2. In some embodiments, the target antigen-binding portion is a Fab or crossover Fab. In some embodiments, the therapeutic protein is mosunetuzumab, glofitamab, cebostamab, or lunimotamab.

[0017] In some embodiments, the bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., a T-cell dependent bispecific antibody; e.g., mosunetuzumab) has a methionine at position 257 of the Fc region (according to the EU index). In some embodiments, the oxidation of the methionine at position 257 of the Fc region is less than 10% over two weeks at 40° C. (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% over two weeks at 40° C.). In some embodiments, the oxidation of the methionine at position 257 of the Fc region is about 6% or less over two weeks at 40° C.

[0018] In another embodiment, the pharmaceutical composition comprises a bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., an anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 bispecific antibody; e.g., TDB or TCB; e.g., anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab), a surfactant (e.g., PS20 or poloxamer 188 (P188)), methionine, and a carrier, wherein the pharmaceutical composition has a pH of about 5.5 or 5.8. In some embodiments, the bispecific antibody comprises at least one target antigen-binding portion and one CD3-binding portion and has a concentration of about 10 mg / ml or less (e.g., 9 mg / ml or less, 8 mg / ml or less, 7.5 mg / ml or less, 7 mg / ml or less, 6 mg / ml or less, 5 mg / ml or less, 4 mg / ml or less, 3 mg / ml or less, 2.5 mg / ml or less, 2 mg / ml or less, 1.5 mg / ml or less, 1.0 mg / ml or less, 0.5 mg / ml or less, 0.25 mg / ml or less, 0.2 mg / ml or less, or 0.1 mg / ml or less, e.g., between 0.01 mg / ml and 0.1 mg / ml). , 0.1 mg / ml to 1 mg / ml, 1 mg / ml to 5 mg / ml, or 5 mg / ml to 10 mg / ml, for example, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, 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 1.5 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml).In some embodiments, the surfactant is at a concentration of 0.05% to 0.12% w / v (e.g., 0.05% to 0.075% or 0.075% to 0.12% 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 0.12% 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 methionine is at a concentration of about 10 mM.

[0019] In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody or a T cell-engaging bispecific antibody; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) 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, 50 to 100, 60 to 80, 70 to 80, or 40 to 50. In certain embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to bispecific antibody is 1 to 100. In some embodiments, the molar ratio of surfactant (e.g., PS20) to bispecific antibody is 45 to 100, 45 to 55, 50 to 100, 60 to 90, 70 to 90, 60 to 80, 70 to 80, 65 to 75, or 75 to 85, e.g., about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In certain embodiments, the molar ratio of surfactant to bispecific antibody is about 71. In another specific embodiment, the molar ratio of surfactant to bispecific antibody is about 79. In some other specific embodiments, the molar ratio of detergent to bispecific antibody is about 48. In some embodiments, the detergent is PS20, and the concentration of PS20 is about 0.06% (w / v).

[0020] In some embodiments, the concentration of the bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is between about 0.1 mg / ml and about 30 mg / ml, e.g., about 0.1 mg / ml and about 10 mg / ml, about 0.1 mg / ml and about 5 mg / ml, about 0.1 mg / ml and about 3 mg / ml, about 0.1 mg / ml and about 2 mg / ml, about 0.1 mg / ml and about 1.5 mg / ml, about 0.3 mg / ml and about 2 mg / ml, about 0.5 mg / ml and about 2 mg / ml, or about 0. 5mg / ml to about 5mg / ml, about 0.5mg / ml to about 2mg / ml, about 0.6mg / ml to about 2mg / ml, about 0.7mg / ml to about 2mg / ml, about 0.8mg / ml to about 2mg / ml, about 0.9mg / ml to about 10mg / ml, about 0.5mg / ml to about 1.4mg / m l, about 0.5mg / ml to about 1.3mg / ml, about 0.5mg / ml to about 1.2mg / ml, about 0.5mg / ml to about 1.1mg / ml, about 0.5mg / ml to about 1.5mg / ml, about 0.8mg / ml to about 1.2mg / ml, 1mg / ml to about 30mg / ml, about 2mg / ml ~30mg / ml, 3mg / ml~30mg / ml, 4mg / ml~30mg / ml, 5mg / ml~30mg / ml, 6mg / ml~30mg / ml, 7mg / ml~30mg / ml, 8mg / ml~30mg / ml, 9mg / ml~30m g / ml, about 10 mg / ml to about 30 mg / ml, about 11 mg / ml to about 30 mg / ml, about 12 mg / ml to about 30 mg / ml, about 13 mg / ml to about 30 mg / ml, about 14 mg / ml to about 30 mg / ml, about 15 mg / ml to about 30 mg / ml, about 20 mg / ml to about 30 mg / ml, approximately 25 mg / ml to approximately 30 mg / ml, approximately 1 mg / ml to approximately 20 mg / ml, approximately 2 mg / ml to approximately 20 mg / ml, approximately 3 mg / ml to approximately 20 mg / ml, approximately 4 mg / ml to approximately 20 mg / ml, approximately 5 mg / ml to approximately 20 mg / ml, approximately 6 mg / ml to approximately 20 mg / m l, about 7 mg / ml to about 20 mg / ml, about 8 mg / ml to about 20 mg / ml, about 9 mg / ml to about 20 mg / ml, about 10 mg / ml to about 20 mg / ml, about 11 mg / ml to about 20 mg / ml, about 12 mg / ml to about 20 mg / ml, about 13 mg / ml to about 20 mg / ml,Approximately 14 mg / ml to approximately 20 mg / ml, approximately 15 mg / ml to approximately 20 mg / ml, approximately 1 mg / ml to approximately 15 mg / ml, approximately 2 mg / ml to approximately 15 mg / ml, approximately 3 mg / ml to approximately 15 mg / ml, approximately 4 mg / ml to approximately 15 mg / ml, approximately 5 mg / ml to approximately 15 mg / ml, approximately 6 mg / ml to approximately 15 mg / ml, approximately 7 mg / ml to approximately 1 5mg / ml, about 8mg / ml to about 15mg / ml, about 9mg / ml to about 15mg / ml, about 10mg / ml to about 15mg / ml, about 11mg / ml to about 15mg / ml, about 12mg / ml to about 15mg / ml, about 12mg / ml to about 14mg / ml, about 13mg / ml to about 15mg / ml, about 13mg / ml to about 14mg / ml, The concentration of the bispecific antibody is about 14 mg / ml to about 15 mg / ml, about 1 mg / ml to about 10 mg / ml, about 2 mg / ml to about 10 mg / ml, about 3 mg / ml to about 10 mg / ml, about 4 mg / ml to about 10 mg / ml, about 5 mg / ml to about 10 mg / ml, about 6 mg / ml to about 10 mg / ml, about 7 mg / ml to about 10 mg / ml, about 8 mg / ml to about 10 mg / ml, about 9 mg / ml to about 10 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.8 mg / ml to about 2.2 mg / ml, about 2 mg / ml to about 5 mg / ml, about 3 mg / ml to about 5 mg / ml, or about 4 mg / ml to about 5 mg / ml. In a specific embodiment, the concentration of the bispecific antibody is about 1 mg / ml. In some embodiments, the concentration of the bispecific antibody is about 3 mg / ml. In some embodiments, the pharmaceutical composition is formulated as a DP.

[0021] In some embodiments, the surfactant is P188, and the concentration of P188 is about 0.08% (w / v). In other embodiments, the molar ratio of surfactant (e.g., P188) to bispecific antibody 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 specific embodiment, the molar ratio of surfactant (e.g., P188) to bispecific antibody is about 14. In another specific embodiment, the molar ratio of surfactant (e.g., P188) to bispecific antibody is about 11.5.

[0022] In some embodiments, the pharmaceutical composition further comprises histidine acetate or histidine HCl at a concentration of about 10 mM or 20 mM and / or sucrose at a concentration of about 240 mM.

[0023] In some embodiments, a bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody) comprises an anti-target arm and an anti-CD3 arm. In some embodiments, the anti-target arm of a bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody) is an anti-CD20 arm, an anti-FcRH5 arm, or an anti-HER2 arm.

[0024] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising hypervariable region (HVR)-H1 comprising the amino acid sequence of SEQ ID NO: 9; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 15; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 16; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 15, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 16. For example, in some embodiments, the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 16, the bispecific antibody is mosunetuzumab.

[0025] In some embodiments, the anti-target arm is an anti-CD20 arm comprising a CD20 binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the CD20-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO:7; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO:8; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD20-binding domain comprises the amino acid sequence of SEQ ID NO:7, and the VL domain of the CD20-binding domain comprises the amino acid sequence of SEQ ID NO:8. For example, in some embodiments, the VH domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO: 7 and the VL domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO: 8, the bispecific antibody is mosunetuzumab.

[0026] In another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody, PS20, and a carrier, wherein the molar ratio of PS20 to bispecific antibody is 100 or less, the PS20 is at a concentration of 0.01% to 0.12% weight / volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-CD20 arm, the anti-CD3 arm comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11, and HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12. The present invention features a pharmaceutical composition comprising a CD3 binding domain comprising HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14; and an anti-CD20 arm comprising a CD20 binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0027] In some embodiments, the CD3-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 15; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 16; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 15, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 16. Additionally or alternatively, in some embodiments, the CD20-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 8; or (c) a VH domain similar to (a) and a VL domain similar to (b). In specific embodiments, the VH domain of the CD20-binding domain comprises the amino acid sequence of SEQ ID NO: 7, and the VL domain of the CD20-binding domain comprises the amino acid sequence of SEQ ID NO: 8. For example, in some embodiments of the pharmaceutical composition, the bispecific antibody is mosunetuzumab.

[0028] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising hypervariable region (HVR)-H1 comprising the amino acid sequence of SEQ ID NO: 65; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 71; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 72; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 71, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 72. For example, in some embodiments, the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 71 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 72, the bispecific antibody is cebostamab.

[0029] In some embodiments, the anti-target arm is an anti-FcRH5 arm comprising an FcRH5 binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62. In some embodiments, the FcRH5-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 63; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 64; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the FcRH5-binding domain comprises the amino acid sequence of SEQ ID NO: 63, and the VL domain of the FcRH5-binding domain comprises the amino acid sequence of SEQ ID NO: 64. For example, in some embodiments, the VH domain of the FcRH5 binding domain comprises the amino acid sequence of SEQ ID NO: 63 and the VL domain of the FcRH5 binding domain comprises the amino acid sequence of SEQ ID NO: 64, the bispecific antibody is cebostamab.

[0030] In another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody, PS20, and a carrier, wherein the molar ratio of PS20 to bispecific antibody is 100 or less, the PS20 is at a concentration of 0.01% to 0.12% weight / volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-FcRH5 arm, the anti-CD3 arm comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 65, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, and HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69. the anti-FcRH5 arm comprises an FcRH5 binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 58, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62.

[0031] In some embodiments, the CD3-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 71; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 72; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 71, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 72. Additionally or alternatively, in some embodiments, the FcRH5-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 63; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 64; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the FcRH5-binding domain comprises the amino acid sequence of SEQ ID NO: 63, and the VL domain of the FcRH5-binding domain comprises the amino acid sequence of SEQ ID NO: 64. For example, in some embodiments of the pharmaceutical composition, the bispecific antibody is cebostamab.

[0032] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising hypervariable region (HVR)-H1 comprising the amino acid sequence of SEQ ID NO: 109; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 111; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 112; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 113; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 114. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 115; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 116; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 115, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 116. For example, in some embodiments, the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 115 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 116, the bispecific antibody is lunimotamab.

[0033] In some embodiments, the anti-target arm is an anti-HER2 arm comprising a HER2 binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 93; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 95; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 96; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 97; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 98. In some embodiments, the HER2-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 99; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 100; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the HER2-binding domain comprises the amino acid sequence of SEQ ID NO: 99, and the VL domain of the HER2-binding domain comprises the amino acid sequence of SEQ ID NO: 100. For example, in some embodiments, the VH domain of the HER2 binding domain comprises the amino acid sequence of SEQ ID NO: 99 and the VL domain of the HER2 binding domain comprises the amino acid sequence of SEQ ID NO: 100, the bispecific antibody is lunimotamab.

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody, PS20, and a carrier, wherein the molar ratio of PS20 to bispecific antibody is 100 or less, the PS20 is at a concentration of 0.01% to 0.12% weight / volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-HER2 arm, and the anti-CD3 arm is composed of HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 111, and HVR-L1 comprising the amino acid sequence of SEQ ID NO: 112. The present invention features a pharmaceutical composition comprising a CD3-binding domain comprising HVR-L2 comprising the amino acid sequence of SEQ ID NO: 113 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 114; and an anti-HER2 arm comprising a HER2-binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 93, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 95, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 96, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 97, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 98.

[0035] In some embodiments, the CD3-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 115; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 116; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 115, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 116. Additionally or alternatively, in some embodiments, the HER2-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 99; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 100; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the HER2-binding domain comprises the amino acid sequence of SEQ ID NO: 99, and the VL domain of the HER2-binding domain comprises the amino acid sequence of SEQ ID NO: 100. For example, in some embodiments of the pharmaceutical composition, the bispecific antibody is lunimotamab.

[0036] In some embodiments, the bispecific antibody comprises an aglycosylation site mutation, e.g., a substitution mutation. In some embodiments, the antibody (e.g., a bispecific antibody, e.g., TDB or TCB) comprises one or more substitution mutations in the Fc region. In certain embodiments, the one or more substitution mutations can reduce the effector function of the bispecific antibody.

[0037] In embodiments where the therapeutic protein is an antibody (e.g., a bispecific antibody, such as TDB or TCB), the antibody can be an IgG antibody (e.g., an IgG1 antibody or an IgG4 antibody). In certain cases, the antibody is a bispecific antibody that is an IgG1 antibody. In some embodiments, the one or more substitution mutations are at one or more amino acid residues selected from the group consisting of N297, L234, L235, D265, and / or P329 (EU numbering). In some embodiments, the one or more substitution mutations in the Fc region comprise one or more knob-in-hole mutations. In some embodiments, the anti-target arm (e.g., an anti-CD20 arm, an anti-FcRH5 arm, or an anti-HER2 arm) comprises T366W and N297G substitution mutations, and the anti-CD3 arm comprises T366S, L368A, Y407V, and N297G substitution mutations.

[0038] At least one of the arms of the bispecific antibody can be monoclonal (e.g., the anti-CD3 arm can be monoclonal, the anti-target arm can be monoclonal, or both the anti-CD3 arm and the anti-target arm can be monoclonal). In some embodiments, at least one of the arms of the bispecific antibody is human, humanized, or chimeric (e.g., the anti-CD3 arm is human, humanized, or chimeric; the anti-target arm is human, humanized, or chimeric; or both the anti-CD3 arm and the anti-target arm are human, humanized, or chimeric).

[0039] In some embodiments, a bispecific antibody (e.g., an anti-CD20 / anti-CD3 bispecific antibody; e.g., a T-cell engaging bispecific antibody) comprises at least one Fab molecule that specifically binds to CD20 comprising the following six hypervariable regions (HVRs): (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, (c) HVR-H3 comprising the amino acid sequence of (SEQ ID NO: 39), (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the bispecific antibody comprises: (a) a heavy chain variable VH domain comprising an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; for example, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 43; (b) a variable light (VL) domain comprising an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; for example, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 44; or (c) at least one Fab molecule that specifically binds to CD20, comprising a VH domain similar to (a) and a VL domain similar to (b). In some embodiments, a Fab molecule that specifically binds to CD20 comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO:43 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:44.

[0040] In some embodiments, the bispecific antibody comprises at least one Fab molecule that specifically binds to CD3, which comprises the following six HVRs: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 45, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46, (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47, (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 48, (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 49, and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the bispecific antibody comprises: (a) a heavy chain variable VH domain comprising an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; for example, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 51; (b) a variable light (VL) domain comprising an amino acid sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; for example, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 52; or (c) at least one Fab molecule that specifically binds CD3, comprising a VH domain similar to (a) and a VL domain similar to (b). In some embodiments, a Fab molecule that specifically binds to CD3 comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO:51 and (b) a VL domain comprising the amino acid sequence of SEQ ID NO:52.

[0041] In another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody, PS20, and a carrier, wherein the molar ratio of PS20 to bispecific antibody is 100 or less, the PS20 is at a concentration of 0.01% to 0.12% weight / volume (w / v), the bispecific antibody comprises one Fab molecule that specifically binds to CD3 and two Fab molecules, each specifically binding to CD20, and the Fab molecules that specifically bind to CD3 are selected from HVR-H1 comprising the amino acid sequence of SEQ ID NO: 45, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47, and HVR-H4 comprising the amino acid sequence of SEQ ID NO: 48. the pharmaceutical composition comprises a CD3-binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42; and two Fabs that specifically bind to CD20 each comprise a CD20-binding domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42.

[0042] In some embodiments, the CD3-binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 51; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 52; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 51, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 52. Additionally or alternatively, in some embodiments, each CD20 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 43; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 44; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of each CD20 binding domain comprises the amino acid sequence of SEQ ID NO: 43, and the VL domain of each CD20 binding domain comprises the amino acid sequence of SEQ ID NO: 44. For example, in some embodiments of the pharmaceutical composition, the bispecific antibody is glofitamab.

[0043] In some embodiments, the bispecific antibody comprises a Fab molecule that specifically binds to CD3, wherein (a) the variable domain of the Fab heavy chain has been swapped with the variable domain of the Fab light chain, or (b) the constant domain of the Fab heavy chain has been swapped with the constant domain of the Fab light chain. In some embodiments, the bispecific antibody comprises at least one Fab molecule that specifically binds to CD20, wherein in the constant domain CL of the Fab molecule, the amino acid at position 124 has been substituted with lysine (K) (Kabat numbering) and the amino acid at position 123 has been substituted with arginine (R) or lysine (K) (Kabat numbering), and in the constant domain CH1 of the Fab molecule, the amino acid at position 147 has been substituted with glutamic acid (E) (EU numbering) and the amino acid at position 213 has been substituted with glutamic acid (E) (EU numbering). In some embodiments, the bispecific antibody is bivalent for CD20 and monovalent for CD3. In some embodiments, the bispecific antibody comprises two Fab molecules that specifically bind to CD20 and one Fab molecule that specifically binds to CD3.

[0044] In some embodiments, the bispecific antibody comprises (a) a first Fab molecule that specifically binds to CD20; (b) a second Fab molecule that specifically binds to CD3; (c) a third Fab molecule that specifically binds to CD20; and (d) an Fc domain consisting of first and second subunits capable of stable association; the third Fab molecule of (c) is identical to the first Fab molecule of (a); and in the constant domain CL of the first Fab molecule of (a) and the third Fab molecule of (c), the amino acid at position 124 is substituted with lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted with arginine (R ) or lysine (K) (Kabat numbering); in the constant domain CH1 of the first Fab molecule of (a) and the third Fab molecule of (c), the amino acid at position 147 is substituted with glutamic acid (E) (EU numbering) and the amino acid at position 213 is substituted with glutamic acid (E) (EU numbering); the first Fab molecule of (a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule of (b), and the second Fab molecule of (b) and the third Fab molecule of (c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of (d).

[0045] In some embodiments, the bispecific antibody is a humanized antibody. In some embodiments, the bispecific antibody is a chimeric antibody.

[0046] In some embodiments, the bispecific antibody comprises an Fc domain, and the Fc domain is an IgG Fc domain. In another embodiment, the IgG Fc domain is an IgG1 Fc domain. In some embodiments, the IgG Fc domain comprises a mutation at amino acid residue N297 (EU numbering) that results in the absence of glycosylation. In some embodiments, the mutation at amino acid residue N297 is a substitution mutation. In some embodiments, the mutation at amino acid residue N297 reduces effector function of the Fc region. In some embodiments, the mutation is an N297G or N297A mutation. In some embodiments, the bispecific antibody comprises a mutation in the Fc region that reduces effector function. In some embodiments, the mutation is a substitution mutation. In some embodiments, the substitution mutation is at amino acid residues L234, L235, D265, and / or P329 (EU numbering). In some embodiments, the mutation is selected from the group consisting of L234A, L235A, D265A, and P329G.

[0047] In some embodiments, the bispecific antibody comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some embodiments, the CH31 domain and the CH32 domain each comprise a protrusion or a cavity, and the protrusion or cavity of the CH31 domain can be positioned, respectively, in the cavity or protrusion of the CH32 domain. In some embodiments, the CH31 and CH32 domains meet at an interface between the protrusion and the cavity. In some embodiments, the CH21 domain and the CH22 domain each comprise a protrusion or a cavity, and the protrusion or cavity of the CH21 domain can be positioned, respectively, in the cavity or protrusion of the CH22 domain. In some embodiments, the CH21 and CH22 domains associate at the interface between the protrusion and the cavity.

[0048] In some embodiments of any of the preceding embodiments, the CD3 binding domain binds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide. The human CD3 polypeptide or the cyno CD3 polypeptide can be, for example, a human CD3ε polypeptide or a cyno CD3ε polypeptide, respectively. Alternatively, the human CD3 polypeptide or the cyno CD3 polypeptide can be a human CD3γ polypeptide or a cyno CD3γ polypeptide, respectively.

[0049] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., 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 some embodiments, the volume of the liquid formulation for dilution is 10 to 20 ml (e.g., 10 to 15 ml, 15 to 20 ml, 13 to 20 ml, 10 to 17 ml, 13 to 17 ml, or 14 to 16 ml; e.g., 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, or about 20 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 15 ml.

[0050] In another specific embodiment, the liquid formulation for dilution is provided in a container having a volume of about 1 ml or about 2 ml (e.g., about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 1.5 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 liquid formulation for dilution is provided in a container having a volume of about 2.5 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 0.5 ml, about 0.9 ml, or about 1 ml.

[0051] In yet another specific embodiment, the liquid formulation for dilution is provided in a container having a volume of about 15 ml (e.g., 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, or about 20 ml). In some embodiments, the liquid formulation for dilution is provided in a container having a volume of about 10 ml. In some embodiments, the volume of the liquid formulation for dilution is 4 to 12 ml (e.g., 4 to 8 ml, 8 to 12 ml, 4 to 10 ml, 6 to 12 ml, 6 to 10 ml, or 7 to 9 ml; e.g., about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, or 12 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 8 ml.

[0052] 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. In some embodiments, the liquid formulation (eg, comprising an anti-FcRH5 / anti-CD3 bispecific antibody; eg, anti-FcRH5 / anti-CD3 TDB; eg, cebostamab) is not diluted.

[0053] 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 in a can (e.g., a mini-can) or a glass container (e.g., a glass vial or glass ampoule).

[0054] 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 particles per ml having a diameter of 2 μm or more). In some embodiments, the carrier is water.

[0055] 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.).

[0056] 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 embodiments, the pharmaceutical composition has a purity of about 95% or greater at about 36 months or longer 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 at about 36 months or longer as assessed by SE-HPLC at about 5° C., e.g., 85%-90%, 90%-95%, or 95%-100% at about 36 months or longer as assessed by SE-HPLC at about 5° C.). In certain embodiments, the pharmaceutical composition has a purity of about 95% or greater at about 42 months or longer as assessed by SE-HPLC at about 5° C., e.g., for about 42 months, about 60 months, about 72 months, about 84 months, about 96 months, or longer at about 5° C.

[0057] 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 as assessed by a non-reducing CE-SDS assay over about 42 months at about 5° C. (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%-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.

[0058] 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)).

[0059] In some embodiments of any of the foregoing aspects and embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intramuscular, topical, oral, transdermal, intraperitoneal, intraorbital, intranasal, intrathecal, or intraventricular administration. For example, in certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In other embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition does not contain a preservative. In some embodiments, the pharmaceutical composition (e.g., comprising an anti-FcRH5 / anti-CD3 bispecific antibody; e.g., anti-FcRH5 / anti-CD3 TDB; e.g., cevostamab) is formulated for administration by infusion without dilution. In some embodiments, the pharmaceutical composition is formulated for administration by infusion 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 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.

[0060] In another aspect, the pharmaceutical composition of any of the foregoing aspects and embodiments is for use as a medicament.

[0061] In another aspect, the pharmaceutical composition of any of the foregoing aspects and embodiments is for use in treating or delaying the progression of a cell proliferative disorder in a subject in need thereof (e.g., a human subject in need thereof).

[0062] 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 a cell proliferative disorder.

[0063] In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the therapeutic protein is a bispecific antibody formulated to bind to a CD3 molecule located on an immune effector cell and to a target molecule located on a target cell other than an immune effector cell (e.g., a CD20 molecule located on (e.g., expressed by) a target cell, such as a B cell). In some embodiments, the bispecific antibody activates the immune effector cell after binding to the CD3 molecule and the target molecule. In some embodiments, the activated immune effector cell can exert a cytotoxic and / or apoptotic effect on the target cell.

[0064] In some embodiments, for example, when the therapeutic protein is a bispecific antibody having an anti-CD20 arm, the cell proliferative disorder is a cancer that 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's lymphoma, Burkitt-like lymphoma with 11q aberration, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), and diffuse large B-cell lymphoma (DLBCL). 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, HHV8-associated multicentric lymphoma Large B-cell lymphoma due to Castleman's disease, 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 lymphoma The cancer is selected from the group consisting of lymphocytic 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 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), or Burkitt's lymphoma (BL).

[0065] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, gastric 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. In some embodiments, the cancer is a HER2-positive cancer.

[0066] In some embodiments, the therapeutic protein is administered in an amount of about 10 μg to about 100 mg (e.g., 100 μg to 80 mg, 500 μg to 50 mg, or 1 mg to 20 mg, e.g., 10 μg to 50 μg, 50 μg to 100 μg, 100 μg to 200 μg, 200 μg to 500 μg, 500 μg to 1 mg, 1 mg to 5 mg, 5 mg to 10 mg, 10 mg to 2 0 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 to 100 mg, for example, about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg , about 100μg, about 200μg, about 250μg, about 300μg, about 400μg, about 500μg, about 600μg, about 700μg, about 750μg, about 800μg, about 900μg , about 1 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, In certain embodiments, the therapeutic protein is a bispecific antibody (e.g., TDB or TCB) formulated for administration to a subject at a dosage of 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 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, or about 100 mg. In certain embodiments, the therapeutic protein is a bispecific antibody formulated for administration to a subject at a dosage of about 1 mg to about 60 mg.

[0067] In some embodiments, the pharmaceutical composition (e.g., comprising an anti-FcRH5 / anti-CD3 bispecific antibody; e.g., anti-FcRH5 / anti-CD3 TDB; e.g., cebostamab) is administered to a subject undiluted (e.g., at a concentration of about 1 mg / ml or about 3 mg / ml). In some embodiments, the pharmaceutical composition is administered to a subject after dilution with saline. In some embodiments, the saline is normal saline. In some embodiments, the saline comprises sodium chloride (NaCl). In some embodiments, the saline solution 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 solution contains 0.45% or 0.9% (w / v) NaCl.

[0068] In some embodiments, after dilution with saline, the concentration of a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is about 0.001 mg / ml to about 0.6 mg / ml (e.g., about 0.001 mg / ml, about 0.002 mg / ml, about 0.003 mg / ml, about 0.004 mg / ml, about 0.005 mg / ml, 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, or about 0.85 mg / ml). 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, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, or about 0.6 mg / ml). In certain embodiments, after dilution with saline, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is about 0.003 mg / ml, about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml. In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TDB; e.g., 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.In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TCB; e.g., glofitamab) is about 0.1 mg / ml or about 0.6 mg / ml. In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-FcRH5 / anti-CD3 bispecific antibody; anti-FcRH5 / anti-CD3 TDB; e.g., cebostamab) is about 0.003 mg / ml, 0.03 mg / ml, or 0.3 mg / ml.

[0069] 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 therapeutic protein is a bispecific antibody comprising an anti-CD3 arm and an anti-CD20 arm, and 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.

[0070] In another aspect, the disclosure features a method of treating or delaying the progression of a cell proliferative disorder 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.

[0071] In another aspect, the disclosure features a method of enhancing immune function in a subject having a cell proliferative disorder, e.g., by administering to the subject an effective amount of the pharmaceutical composition of any of the preceding aspects.

[0072] In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the therapeutic protein of the pharmaceutical composition administered to a subject is a bispecific antibody formulated to bind to a CD3 molecule located on an immune effector cell and to a target molecule located on a target cell other than an immune effector cell (e.g., a CD20 molecule located on (e.g., expressed by) a target cell, such as a B cell). In some embodiments, the bispecific antibody activates the immune effector cell after binding to the CD3 molecule and the target molecule. In some embodiments, the activated immune effector cell can exert a cytotoxic and / or apoptotic effect on the target cell.

[0073] In some embodiments, for example, when the therapeutic protein is a bispecific antibody having an anti-CD20 arm, the cell proliferative disorder is a cancer that 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's lymphoma, Burkitt-like lymphoma with 11q aberration, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), and diffuse large B-cell lymphoma (DLBCL). 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, HHV8-associated multicentric lymphoma Large B-cell lymphoma due to Castleman's disease, 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 lymphoma The cancer is selected from the group consisting of lymphocytic 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 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), or Burkitt's lymphoma (BL).

[0074] In some embodiments, the cancer is selected from the group consisting of breast cancer, colorectal cancer, gastric 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. In some embodiments, the cancer is a HER2-positive cancer.

[0075] In some embodiments, the therapeutic protein (e.g., bispecific antibody) is administered in an amount of about 10 μg to about 100 mg (e.g., 100 μg to 80 mg, 500 μg to 50 mg, or 1 mg to 20 mg, e.g., 10 μg to 50 μg, 50 μg to 100 μg, 100 μg to 200 μg, 200 μg to 500 μg, 500 μg to 1 mg, 1 mg to 5 mg, 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 30 mg, 30 mg to 40 mg, 40 mg to 50 mg, 50 mg to 60 mg, 60 mg to 70 mg, 70 mg to 80 mg, 80 mg to 90 mg, or 90 to 100 mg, e.g., about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, Approximately 70μg, approximately 75μg, approximately 80μg, approximately 90μg, approximately 100μg, approximately 200μg, approximately 250μg, approximately 300μg, approximately 400μg, approximately 500μg, approximately 600μg, approximately 700μg, about 750μg, about 800μg, about 900μg, about 1mg, about 2mg, about 2.5mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, In certain embodiments, the method comprises administering to a subject a therapeutic protein (e.g., a bispecific antibody) at a dosage of about 1 mg to about 60 mg.

[0076] 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 therapeutic protein is a bispecific antibody comprising an anti-CD3 arm and an anti-CD20 arm, and the at least one additional therapeutic agent is an ADC (e.g., an anti-CD79b In some embodiments, the pharmaceutical composition comprises an ADC (e.g., polatuzumab vedotin). In some embodiments, the pharmaceutical composition is administered by intravenous, subcutaneous, intramuscular, topical, oral, transdermal, intraperitoneal, intraorbital, intranasal, intrathecal, or intracerebroventricular administration. In some embodiments, the subject is a human.

[0077] Any and all embodiments may be combined unless the context clearly indicates otherwise. Any and all embodiments may be applied to any and all aspects of this disclosure unless the context clearly indicates otherwise.

[0078] 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]

[0079] [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 IV bag shaking studies in 100 ml PO bags. 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 anti-CD20 / anti-CD3 TDB 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 TDB, the second bar represents 2 mg TDB, and the third bar represents 1 mg TDB. [Figure 4A] Figure 4A is a graph showing the kinetics of protein oxidation at methionine 257 (Met257) of mosunetuzumab 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] Figure 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 for 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 compositions stored at 5°C for up to 12 months. BTCT4465A 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 arm of 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 5. [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 5. [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 5. [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 5. [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 5. [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 5. [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 5. [Figure 11B] Figure 11B is a graph showing the change over time in the sum of the main peaks 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 5. [Figure 12] FIG. 12 is a graph showing the Donnan effect on pH values ​​of mosunetuzumab compositions. [Figure 13] Schematic diagram showing the structure of glofitamab. [Figure 14] Formulation development GLP Tox and introduction into human studies. Surfactant content of formulations F1 to F5, initially compared to after 6 weeks of storage at 5, 25, or 40°C. [Figures 15A-15C] Formulation development GLP Tox and introduction into human studies, size exclusion chromatography (SEC) of formulations F1 to F5, initial and after 6 weeks of storage at 5, 25 or 40°C. Figure 15A: Main peak; Figure 15B: High molecular weight (HMW); Figure 15C: Low molecular weight (LMW). [Figures 16A-16C]Formulation Development GLP Tox and Implementation into Human Studies, Ion Exchange Chromatography (IEC) of Formulations F1 to F5, Initial and After 6 Weeks of Storage at 5, 25, or 40°C. Figure 16A: Main Peak; Figure 16B: HMW; Figure 16C: LMW [Figure 17] Formulation Development—Analysis of Formulation F1 through Week 84. F1=5 mg / ml RO7022859 (i.e., glofitamab), 20 mM histidine-HCl pH 5.5, 240 mM sucrose, 10 mM methionine, 0.05% (w / v) polysorbate 20. [Figure 18A] Formulation development GLP Tox and introduction into human studies, huCD20 binding of formulations F1 to F5, initially compared with after 3 and 6 weeks of storage at 5, 25, or 40°C. [Figure 18B] Formulation development GLP Tox and introduction into human studies, huCD3 binding of formulations F1 to F5, initially compared with after 3 and 6 weeks of storage at 5, 25, or 40°C. [Figures 19A-19B] Development studies for Phase III and commercial formulation. Size exclusion (SE)-HPLC % HMWS (Figure 19A) and ion exchange (IE)-HPLC % acidic area (Figure 19B) of glofitamab as a function of protein concentration after 104 weeks of storage at 5°C. [Figures 20A-20B] Development studies for Phase III and commercial formulation. Glofitamab SE-HPLC % HMWS (Figure 20A) and IE-HPLC % acidic area (Figure 20B) as a function of pH and stabilizer (methionine) addition after 6 weeks of storage at 40°C. [Figure 21] Development studies for Phase III and commercial formulation. SE-HPLC % HMWS and IE-HPLC % acidic area of ​​glofitamab, including visible particle formation, as a function of tonicity agent after 26 weeks of storage at 25°C. [Figure 22] Development studies for Phase III and commercial formulation. SE-HPLC % HMWS and IE-HPLC % acidic area of ​​glofitamab including visible particle formation as a function of surfactant after 7 days of shaking at 25°C. [Figure 23]Development study for phase III and commercial formulation. Glofitamab PS20 content [mg / ml] and visible particle formation as a function of protein concentration initially and after 104 weeks of storage at 5°C. [Figure 24] Long-term stability data: PS20 content of example glofitamab DP batches with respect to stability (storage at 2-8°C). DETAILED DESCRIPTION OF THE INVENTION

[0080] DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS 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.

[0081] The term "about," as used herein, refers to a normal range of error for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0082] 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.

[0083] 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.

[0084] The terms "pharmaceutical composition" or "pharmaceutical formulation" are used interchangeably herein and refer to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0085] The term "therapeutic protein" refers to a protein that can be administered to a subject to induce a biological response associated with the treatment or amelioration of a disease or pathological condition. Therapeutic proteins include biological drugs, such as peptides, antibodies (e.g., bispecific antibodies, e.g., T cell-dependent bispecific antibodies (TDBs), e.g., anti-CD20 / anti-CD3 TDBs, e.g., mosunetuzumab, T cell-engaging bispecific antibodies (TCBs), e.g., anti-CD20 / anti-CD3 TCBs, e.g., glofitamab), and immunoconjugates.

[0086] 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.

[0087] The term "shelf life" refers to the length of time that a product (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB, e.g., anti-CD20 / anti-CD3 TDB, e.g., mosunetuzumab, e.g., TCB, e.g., anti-CD20 / anti-CD3 TCB, e.g., glofitamab)) 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.

[0088] A "stable" pharmaceutical formulation is one in which the protein (e.g., a therapeutic protein) therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Preferably, the formulation essentially retains its physical and chemical stability and its biological activity upon storage (e.g., upon 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, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., 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 can 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 of the protein (e.g., Met residues of antibodies); 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 (e.g., therapeutic proteins); 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 TDB 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.

[0089] A protein (e.g., a therapeutic protein, e.g., a bispecific antibody) "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.

[0090] A protein (e.g., a therapeutic protein such as a bispecific antibody) "retains its chemical stability" in a pharmaceutical formulation if its chemical stability at a given time is such that the protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB)) is believed to still retain its biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of a protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB, e.g., anti-CD20 / anti-CD3 TDB or TCB)). Chemical alterations can include protein oxidation, which can be assessed using, for example, tryptic peptide mapping, reverse-phase high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC / MS). Other types of chemical alterations include charge alterations of a protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB, e.g., anti-CD20 / anti-CD3 TDB or TCB)), which can be assessed, for example, by ion exchange chromatography or icIEF.

[0091] A protein (e.g., a therapeutic protein) "retains its biological activity" in a pharmaceutical formulation if the biological activity of the protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB)) 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.

[0092] As used herein, "biological activity" of a protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB)) refers to the ability of the protein (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., TDB or TCB)) to bind to its target, e.g., the ability of an antibody to bind to its antigen (e.g., the ability of TDB or TCB 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.

[0093] An "oxidation-susceptible" protein (e.g., a therapeutic protein, e.g., a bispecific antibody, e.g., TDB or TCB) 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 a therapeutic protein such as TDB or TCB may be susceptible to oxidation.

[0094] The term "percent oxidation" refers to the percent of a protein (e.g., a therapeutic protein) in a formulation (e.g., a 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., a therapeutic protein) or pharmaceutical composition thereof.

[0095] As used herein, the term "assessed by AAPH stress test" means that the percent oxidation at a specific amino acid residue (e.g., Met residue) is determined by mass spectrometry of tryptic peptides after formulating the protein (e.g., a therapeutic protein, such as TDB or TCB) 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 of, for example, about 10 mg / ml of therapeutic protein, about 10 mM histidine acetate, about 240 mM sucrose, about 0.06 (w / v) polysorbate 20, pH about 5.8. The stressed protein (e.g., a therapeutic protein, such as TDB or TCB) is digested with trypsin, and the digested peptides are subjected to LC-MS-MS to determine the percentage of oxidation.

[0096] As used herein, "buffer" refers to a buffer solution (also referred to herein as a "buffering agent") that resists changes in pH through the action of its acid-base conjugate 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 5.1 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 5.5 or 5.8. In certain embodiments, the buffer solution is about pH 5.5. In another specific embodiment, the buffer solution is about pH 5.8. Exemplary buffering agents for use in the present disclosure include, but are not limited to, histidine (e.g., histidine acetate or histidine hydrochloride (HCl)), acetate, phosphate, succinate, or a combination thereof. In some embodiments, the histidine is histidine acetate or histidine HCl, monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or a mixture thereof. In certain embodiments, the buffering agent is histidine HCl or histidine acetate.

[0097] As used herein, "tonicity 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, the tonicity 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, the tonicity agent is used to reduce local irritation by preventing osmotic shock at the application site (e.g., during subcutaneous administration). Exemplary tonicity agents include carbohydrates (e.g., sucrose, glucose, dextrose, glycerol, glycerin, mannitol, and trehalose), amino acids, and salts (e.g., sodium chloride and potassium chloride).

[0098] 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).

[0099] 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 group is a long-chain compound), 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.

[0100] The "molar ratio of detergent to therapeutic protein" (detergent:protein) is the ratio of detergent to therapeutic protein, with each component expressed in molar concentration (also called molarity). Equation (1) shows this ratio: TIFF2025512342000001.tif17170

[0101] As used herein, "drug substance" or "DS" refers to a pharmaceutical composition formulated for storage, for example, frozen storage, before administration to a subject. The DS may have a higher concentration of a therapeutic protein than the concentration of the therapeutic protein administered to a subject. Therefore, in some instances, the DS is diluted before administration to a subject.

[0102] As used herein, "pharmaceutical product" or "DP" refers to a pharmaceutical composition in its final configuration (e.g., in final vial configuration) as ready to be administered to a subject. The concentration of a therapeutic protein in a DP may be the concentration administered to a subject. Alternatively, if the DP is intended to be administered with a diluent or in combination with other therapeutic agents, the DP may be at a higher concentration than that administered to a subject.

[0103] In this application, unless otherwise specified, the techniques utilized can 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).

[0104] 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.

[0105] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids, not a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides may arise in any manner, including by chemical synthesis. Polypeptides of the present disclosure may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids. Polypeptides can have a defined three-dimensional structure, but polypeptides do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but rather can adopt a number of different conformations are referred to as unfolded.

[0106] By "isolated" polypeptide or variant or derivative thereof is intended a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0107] 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., bispecific antibodies, such as TDB or TCB), and antibody fragments, so long as they exhibit the desired antigen-binding activity (e.g., an antigen-binding fragment of an antibody).

[0108] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives, such as fragments thereof.

[0109] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an 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'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0110] "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, and the VH and / or VL domains of an antibody), a receptor, a ligand, an aptamer, or other molecule with an identified binding partner.

[0111] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target an entity to which it is bound (e.g., a cytokine or a second antigen-binding moiety) to a target site, such as a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof, as further defined herein. Preferred antigen-binding moieties include the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, antigen-binding moieties comprise antibody constant regions known in the art, as further defined below. Useful heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of two isotypes: κ and λ.

[0112] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of a Fab heavy chain have been swapped (i.e., replaced with one another), i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, N-terminal to C-terminal), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N-terminal to C-terminal). For clarity, in a crossover Fab molecule in which the variable domains of a Fab light chain and a Fab heavy chain have been swapped, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of a Fab light chain and a Fab heavy chain have been swapped, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0113] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain consisting of the heavy chain variable and constant domains (from N- to C-terminus: VH-CH1) and a light chain consisting of the light chain variable and constant domains (from N- to C-terminus: VL-CL).

[0114] 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 that form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contacts"). Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs of the invention 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).

[0115] 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.

[0116] 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 native antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6 th(See, e.g., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using the VH or VL domain of an antibody that binds the antigen to screen 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).

[0117] 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, the 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.

[0118] "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 VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0119] "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 to achieve the maximum percent sequence identity and introducing gaps, if necessary, 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 across the entire 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.

[0120] 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 relative to a given amino acid sequence B (alternatively, it may be written as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given 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, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0121] 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.

[0122] 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 as described in Kabat et al., supra.

[0123] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from 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 a non-human antibody and all or substantially all of the FRs correspond to 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.

[0124] 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.

[0125] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin using human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Human antibodies can be generated using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using 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). 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 have their endogenous gene locus disabled (e.g., XENOMOUSE (商標) (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related 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.

[0126] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible minor variant antibodies, including, for example, naturally occurring mutations or mutations that arise during the production of monoclonal antibody preparations. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of 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.

[0127] 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.

[0128] "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 disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a 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 contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a 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) or lambda (λ).

[0129] As used herein, the term "half antibody" refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain.

[0130] 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).

[0131] 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 corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0132] 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 cyno CD3ε protein (NCBI RefSeq No. NP_001270544.1), and the 181 amino acid long cynomolgus monkey CD3γ protein (NCBI RefSeq No. NP_001270839.1).

[0133] 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 and any form of CD20 resulting from intracellular processing. 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 can be produced from a variant mRNA transcript that is, for example, 297 amino acids in length and that lacks, for example, a portion of the 5'UTR (see, e.g., NCBI Reference SEQ ID NO: NM_021950.3), or a longer variant mRNA transcript (see, e.g., NCBI Reference SEQ ID NO: NM_152866.2).

[0134] As used herein, the term "FcRH5" or "fragment crystalline receptor-like 5" refers to any native FcRH5 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified, and includes "full-length," unprocessed FcRH5, as well as any form of FcRH5 resulting from intracellular processing. This term also encompasses naturally occurring variants of FcRH5, including, for example, splice variants or allelic variants. FcRH5 includes, for example, the human FcRH5 protein (UniProtKB / Swiss-Prot ID: Q96RD9.3), which is 977 amino acids long.

[0135] The term "HER2," as used herein, unless otherwise indicated, refers to any native HER2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses not only "full-length" unprocessed HER2, but also any form of HER2 resulting from intracellular processing. The term also encompasses naturally occurring variants of HER2, including, for example, splice variants or allelic variants. HER2 includes, for example, the human HER2 protein, which is 1240 amino acids long (see, for example, NCBI RefSeq No. NP_001276865). Domain IV of HER2 is the extracellular protein region closest to the cell membrane. Domain IV has the amino acid sequence of SEQ ID NO: 17.

[0136] The terms "anti-CD20 / anti-CD3 antibody," "anti-CD20 / anti-CD3 antibody," "anti-CD20 / anti-CD3 TDB," and "anti-CD20 / anti-CD3 TCB," 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 an unrelated non-CD3 protein and / or a non-CD20 protein 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, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (K D) binds to CD20 and / or CD3, respectively. In certain embodiments, the anti-CD20 / anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species and / or an epitope of CD20 that is conserved among CD20 from different species. Anti-CD20 / anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-CD20 / anti-CD3 bispecific antibodies described in PCT Publication No. 2015 / 095392, which is incorporated herein by reference in its entirety. In some examples, the anti-CD20 / anti-CD3 bispecific antibody is an anti-CD20 / anti-CD3 T-cell-dependent bispecific antibody (TDB). In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab (also known as BTCT4465A or RG 7828), as defined by International Nonproprietary Names for Pharmaceutical Substances (INN) List 117 (WHO Drug Information, Vol. 31, No. 2, 2017, pp. 304-305). In other embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a T-cell activating bispecific antigen binding molecule (e.g., a 2+1 TCB) described in U.S. Pat. No. 9,914,776, the entire contents of which are incorporated herein by reference. In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab (also known as RO 7082859 or RG6026), as defined by the International Nonproprietary Names for Pharmaceutical Substances (INN) List 121 (WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN:List 83, 2020, vol. 34, no. 1, p. 39), CAS Registry Number 2229047-91-8.

[0137] The terms "anti-FcRH5 / anti-CD3 antibody," "anti-FcRH5 / anti-CD3 bispecific antibody," and "anti-FcRH5 / anti-CD3 TDB," or variants thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that can bind to FcRH5 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting FcRH5 and / or CD3. In one embodiment, the extent of binding of an anti-FcRH5 / anti-CD3 bispecific antibody to unrelated non-CD3 and / or non-FcRH5 proteins is less than about 10% of the binding of the antibody to CD3 and / or FcRH5, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an anti-FcRH5 / anti-CD3 bispecific antibody has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (K D) binds to FcRH5 and / or CD3, respectively. In certain embodiments, the anti-FcRH5 / anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species and / or an epitope of FcRH5 that is conserved among FcRH5 from different species. Anti-FcRH5 / anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-FcRH5 / anti-CD3 bispecific antibodies described in PCT Publication Nos. WO2016 / 205520, WO2015 / 095392, and WO2014 / 210064, and U.S. Pat. No. 10,323,094, the entire contents of which are incorporated herein by reference. In some examples, the anti-FcRH5 / anti-CD3 bispecific antibody is an anti-FcRH5 / anti-CD3 T cell-dependent bispecific antibody. In a specific embodiment, the anti-FcRH5 antibody is cebostamab (also known as BFCR4350A or RO7187797), which is described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN:List 84, Vol. 34, No. 3, published 2020 (see page 701), and has CAS Registry Number 2249888-53-5. Sebostamab is an Fc-engineered humanized full-length aglycosylated IgG1κ T-cell-dependent bispecific antibody (TDB) that binds to FcRH5 and CD3.

[0138] The terms "anti-HER2 / anti-CD3 antibody," "anti-HER2 / anti-CD3 bispecific antibody," and "anti-HER2 / anti-CD3 TDB," or variants thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that can bind to HER2 and CD3 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting HER2 and / or CD3. In one embodiment, the extent of binding of an anti-HER2 / anti-CD3 bispecific antibody to unrelated non-CD3 and / or non-HER2 proteins is less than about 10% of the binding of the antibody to CD3 and / or HER2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an anti-HER2 / anti-CD3 bispecific antibody has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (K D) binds to HER2 and / or CD3, respectively. In certain embodiments, the anti-HER2 / anti-CD3 antibody binds to an epitope on CD3 that is conserved among CD3 from different species and / or an epitope on HER2 that is conserved among HER2 from different species. Anti-HER2 / anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-HER2 / anti-CD3 bispecific antibodies described in PCT Publication No. 2015 / 095392, which is incorporated herein by reference in its entirety. In some examples, the anti-HER2 / anti-CD3 bispecific antibody is an anti-HER2 / anti-CD3 T-cell-dependent bispecific antibody. In a specific embodiment, the anti-HER2 / anti-CD3 bispecific antibody (anti-HER2 / anti-CD3 TDB) is lunimotamab, as defined by the International Nonproprietary Names for Pharmaceutical Substances (INN) List 124 (WHO Drug Information, Vol. 34, No. 4, 2020, p. 1031), and has CAS Registry Number 2361325-98-4.

[0139] As used herein, the term "cluster of differentiation 79b" or "CD79b," unless otherwise indicated, refers to any native CD79b 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 CD79b, as well as any form of CD79b resulting from intracellular processing. The term also encompasses naturally occurring variants of CD79b, including, for example, splice variants or allelic variants. CD79b includes, for example, the human CD79b protein (NCBI RefSeq No. NP_000617), which is 229 amino acids in length.

[0140] The terms "anti-CD79b antibody" and "antibody that binds to CD79b" refer to an antibody that is capable of binding to CD79b with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD79b. In one embodiment, the binding of an anti-CD79b antibody to an unrelated, non-CD79b protein is less than about 10% of the binding of the antibody to CD79b as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD79b 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 In certain embodiments, the anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79b from different species.

[0141] As used herein, the term "polatuzumab vedotin" refers to an anti-CD79b antibody-drug conjugate having IUPHAR / BPS number 8404, KEGG number D10761, or CAS registry number 1313206-42-6. Polatuzumab vedotin-piiq is also referred to interchangeably as "polatuzumab vedotin piiq," "huMA79bv28-MC-vc-PAB-MMAE," "DCDS4501A," or "RG7596."

[0142] In some embodiments, an antibody drug conjugate is used that comprises the following formula: TIFF2025512342000002.tif24170, where Ab is polatuzumab and p is 1 to 8. In some embodiments, the anti-CD79b antibody drug conjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the antibody drug conjugate is polatuzumab vedotin-piiq (CAS Registry Number 1313206-42-6).

[0143] In some embodiments, the antibody drug conjugate is polatuzumab vedotin-piiiq, as described in WHO Drug Information, Vol. 26, No. 4, 2012 (Proposed INN: List 108), which is expressly incorporated herein by reference in its entirety. As shown in WHO Drug Information, Vol. 26, No. 4, 2012, polatuzumab vedotin-piiq has the following structure: immunoglobulin G1-kappa auristatin E conjugate, anti-[Homo sapiens CD79B (immunoglobulin-related CD79β)], ​​a humanized monoclonal antibody conjugated to auristatin E; gamma 1 heavy chain (1-447) [humanized VH(Homo sapiens IGHV3-66*01(79.60%)-(IGHD)-IGHJ4*01)[8.8.13](1-120)-Homo sapiens IGH1*03(CH1 R120>K(214)(121–218), hinge(219–233), CH2(234–343), CH3(344–448), CHS(449–450))(121–450)], (220–218′)-disulfide (when unconjugated) and kappa light chain(1′–218′) [humanized V-KAPPA (Homo sapiens IGKV1-39*01(80.00%)-IGKJ1*01] [11.3.9 ](1'-112')-Homo sapiens IGKC*01(113'-218')]; dimeric (226-226":229-229")-bisdisulfide; conjugated with an average of 3-4 cysteinyls to monomethyl auristatin E (MMAE) via a cleavable maleimidocaproyl-valyl-citrullinyl-p-aminobenzylcarbamate (mc-val-cit-PABC) linker.

[0144] A "subject" or "individual" is a mammal. Mammals include, but are not limited to, livestock 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.

[0145] As used herein, "administering" refers to a method of providing a subject with a dosage of a therapeutic protein (e.g., TDB or TCB) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a therapeutic protein (e.g., TDB or TCB) of the present disclosure). Pharmaceutical compositions utilized in the methods described herein can be administered, for example, intravenously, subcutaneously, intradermally, intramuscularly, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraumbilically, intraocularly, intrabuccally, locally, topically, by inhalation, by injection, by infusion, by continuous infusion, by localized bath target cell, directly, by catheter, by perfusion, in a cream, or in a lipid composition. The method of administration can vary depending on various factors (e.g., the pharmaceutical composition being administered and the severity of the symptom, disease, or disorder being treated).

[0146] As used herein, a "week" is 7 days ± 2 days.

[0147] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be carried out prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and improving or improving 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.

[0148] 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 known to those of skill in the art.

[0149] 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, and 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%. Methods for measuring this enhancement are known to those skilled in the art.

[0150] As used herein, "delaying the progression" of a disorder or disease means postponing, preventing, slowing down, retarding, stabilizing, and / or delaying the development of a disease or disorder (e.g., a cell proliferative disorder, e.g., cancer). This delay can be of various durations depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the development of metastasis, can be delayed.

[0151] "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 can refer to antibody effector functions mediated by the antibody Fc region, such effector functions specifically including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0152] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0153] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

[0154] 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 squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, or stomach cancer. cancer), such as gastrointestinal and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade These include, but are not limited to, diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved 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 abnormal blood vessel proliferation associated with phacomatosis, edema (such as that associated with brain tumors), Meigs' syndrome, brain cancer, and head and neck cancer, and associated metastases. In certain embodiments, cancers amenable to treatment with the antibodies of the present disclosure include breast cancer, colorectal cancer, gastric cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Further, in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, gastric cancer, glioblastoma, and breast carcinoma (including metastatic forms of these cancers). In other embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, 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 variant, heavy chain disease, alpha heavy chain disease, Selected from mature B-cell cancers including 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 of the CNS, 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, plasmablastic 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.

[0155] The term "HER2-positive" cancer includes cancer cells that have more HER2 than normal. Examples of HER2-positive cancer include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, the HER2-positive cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification of ≧2.0. In some examples, the HER2-positive cancer is a HER2-positive solid tumor. Additionally or alternatively, the HER2-positive cancer can be a locally advanced or metastatic HER2-positive cancer. In some examples, the HER2-positive cancer is a HER2-positive breast cancer or a HER2-positive gastric cancer. In some embodiments, the HER2-positive cancer is selected from the group consisting of HER2-positive gastroesophageal junction cancer, HER2-positive colorectal cancer, HER2-positive lung cancer (e.g., HER2-positive non-small cell lung carcinoma), HER2-positive pancreatic cancer, HER2-positive bladder cancer, HER2-positive salivary duct cancer, HER2-positive ovarian cancer (e.g., HER2-positive epithelial ovarian cancer), or HER2-positive endometrial cancer.

[0156] The term "FcRH5-positive cancer" refers to a cancer containing cells expressing FcRH5 on their surface. For purposes of determining whether a cell expresses FcRH5 on its surface, FcRH5 mRNA expression is considered to correlate with FcRH5 expression on the cell surface. In some embodiments, FcRH5 mRNA expression is determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR). Alternatively, FcRH5 expression on the cell surface can be determined using an antibody against FcRH5 by methods such as immunohistochemistry, FACS, etc. In some embodiments, FcRH5 is one or more of FcRH5a, FcRH5b, FcRH5c, UniProt identifier Q96RD9-2, and / or FcRH5d. In some embodiments, FcRH5 is FcRH5c. Specific examples of FcRH5-positive cancers include FcRH5-positive multiple myeloma (MM), FcRH5-positive chronic lymphocytic leukemia (CLL), FcRH5-positive mantle cell lymphoma (MCL), FcRH5-positive diffuse large B-cell lymphoma (DLBCL), FcRH5-positive follicular lymphoma (FL), FcRH5-positive acute myeloid leukemia (AML), FcRH5-positive myelodysplastic syndrome (MDS), FcRH5-positive chronic myelogenous leukemia (CML), FcRH5-positive chronic myelomonocytic leukemia, and FcRH5-positive leukemia. These include FcRH5-positive acute promyelocytic leukemia (APL), FcRH5-positive chronic myeloproliferative disorder, FcRH5-positive thrombocytopenic leukemia, FcRH5-positive precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), FcRH5-positive precursor T-cell acute lymphoblastic leukemia (pre-T-ALL), FcRH5-positive obesity disease, FcRH5-positive mast cell leukemia, FcRH5-positive mast cell sarcoma, FcRH5-positive myeloid sarcoma, FcRH5-positive lymphocytic leukemia, and FcRH5-positive undifferentiated leukemia.

[0157] 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.

[0158] 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 can only be presented 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 in tumor cells due to the structure of tumor tissue being smaller than that of normal tissue.

[0159] "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.

[0160] As used herein, the term "effector cells" refers to a population of lymphocytes that express effector moiety receptors, e.g., cytokine receptors, and / or Fc receptors, on their surface, thereby binding effector moieties, e.g., cytokines, and / or the Fc region of antibodies, and contributing to the destruction of target cells, e.g., tumor cells. Effector cells mediate, for example, cytotoxic or phagocytic effects. Effector cells include CD8 + cytotoxic T cells, CD4 + These include, but are not limited to, effector T cells such as helper T cells, γδ T cells, NK cells, lymphocyte-activated killer (LAK) cells, and macrophages / monocytes.

[0161] A "modification that promotes association of the first and second Fc domain subunits" refers to a manipulation of the peptide backbone or a post-translational modification of the Fc domain subunit that reduces or prevents the association of a peptide containing the Fc domain subunit with an identical polypeptide to form homodimers. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second Fc domain subunits) that are desired to associate, where these modifications are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of the Fc domain subunits to sterically or electrostatically favor their association, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, the modification that promotes association comprises an amino acid mutation, particularly an amino acid substitution, within the Fc domain, hi certain embodiments, the modification that promotes association comprises a separate amino acid mutation, particularly an amino acid substitution, in each of the two subunits of the Fc domain.

[0162] An "activating Fc receptor" is an Fc receptor that, following engagement by the Fc region of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0163] As used herein, the terms "engineer," "engineered," and "engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes altering the amino acid sequence, altering the glycosylation pattern, or altering the side groups of individual amino acids, and combinations of these approaches. In particular, "engineering" with the prefix "glyco," and the term "glycosylation engineering" include metabolic engineering of the glycosylation machinery of a cell, including genetic manipulation of the oligosaccharide synthesis pathway to achieve altered glycosylation of glycoproteins expressed in the cell. Furthermore, glycosylation engineering includes the influence of mutations and the cellular environment on glycosylation. In one embodiment, glycosylation engineering is alteration of glycosyltransferase activity. In certain embodiments, engineering results in altered glucosaminyltransferase activity and / or fucosyltransferase activity. Glycosylation engineering can be used to obtain "host cells with increased GnTIII activity" (e.g., host cells engineered to have increased expression levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), "host cells with increased ManII activity" (e.g., host cells engineered to have increased expression levels of one or more polypeptides having α-mannosidase II (ManII) activity), or "host cells with reduced α(1,6)fucosyltransferase activity" (e.g., host cells engineered to have reduced expression levels of α(1,6)fucosyltransferase).

[0164] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original transformed cell and progeny derived from the original transformed cell, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Included herein are progeny of mutants that have the same function or biological activity as screened or selected for in the originally transformed cell. Host cells are any type of cell line that can be used to produce proteins used in the present disclosure. In one embodiment, the host cells are engineered to enable the production of antibodies with modified oligosaccharides. In certain embodiments, the host cells are engineered to increase the expression level of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cells are further engineered to increase the expression level of one or more polypeptides having α-mannosidase II (ManII) activity. Host cells include cultured cells, e.g., cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse bone marrow cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0165] As used herein, the term "polypeptide having GnTIII activity" refers to a polypeptide capable of catalyzing the addition of an N-acetylglucosamine (GlcNAc) residue in a β-1,4 linkage to the β-linked mannoside of a trimannosyl core of an N-linked oligosaccharide. This includes fusion polypeptides that exhibit enzymatic activity similar, but not necessarily identical, to the activity of β(1,4)-N-acetylglucosaminyltransferase III, also known as β-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC 2.4.1.144) according to the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Commission on Nomenclature, as measured in specific biological assays, with or without dose dependency. If a dose dependency exists, it need not be identical to that of GnTIII, but rather should be substantially similar to the dose dependency for a given activity when compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or at least about 25-fold, preferably at least about 10-fold, and most preferably at least about 3-fold less activity than GnTIII). In certain embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi-resident polypeptide. In particular, the Golgi localization domain is the localization domain of mannosidase II or GnTI, most particularly the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of α1,6 core fucosyltransferase. Methods for making such fusion polypeptides and using them to generate antibodies with increased effector function are disclosed in WO 2004 / 065540, U.S. Provisional Patent No. 60 / 495142, and U.S. Patent Publication No. 2004 / 0241817, the entire contents of which are expressly incorporated herein by reference.

[0166] As used herein, the term "Golgi localization domain" refers to an amino acid sequence of a Golgi-resident polypeptide that is responsible for anchoring the polypeptide to a location within the Golgi complex. Generally, the localization domain constitutes the amino-terminal "tail" of the enzyme.

[0167] As used herein, the term "polypeptide having ManII activity" refers to a polypeptide capable of catalyzing the hydrolysis of terminal 1,3- and 1,6-linked α-D-mannose residues of the branched GlcNAcMan5GlcNAc2 mannose intermediate of N-linked oligosaccharides. This includes polypeptides that exhibit activity similar, but not necessarily identical, to that of Golgi α-mannosidase II, also known as mannosyloligosaccharide 1,3-1,6-α-mannosidase II (EC 3.2.1.114) according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).

[0168] An "effective amount" of a pharmaceutical composition, e.g., a pharmaceutical composition comprising a therapeutic protein, is at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, such as a measurable improvement of a particular disorder (e.g., a cell proliferative disorder, e.g., 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 the individual. An effective amount is also one in which any toxic or adverse effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as a reduction in the risk, severity, or delay in 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 another drug 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 halting) cancer cell invasion into peripheral organs, inhibiting (i.e., slowing or desirably halting) tumor metastasis, inhibiting tumor growth, and / or alleviating 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 the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0169] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, including information regarding the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings regarding such therapeutic product.

[0170] 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 so as 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.

[0171] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, impairs, or prevents signaling that occurs as a result of the interaction of PD-1 with one or more of its 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 certain aspects, 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, impair, or prevent signaling that results 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 mediated PD-1-mediated signaling, rendering dysfunctional T cells less dysfunctional (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.

[0172] 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 negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate PD-L1-mediated signaling, alleviating the dysfunctional state of dysfunctional T cells (e.g., enhancing the effector response 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.

[0173] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., 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 particular aspects, 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 resulting 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 on T lymphocytes that mediate signaling through PD-L2, reducing 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.

[0174] II. Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising therapeutic proteins, e.g., bispecific antibodies (e.g., anti-CD3 bispecific antibodies; e.g., anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 bispecific antibodies; e.g., T cell-dependent bispecific antibodies (TDBs) or T cell-engaging bispecific antibodies (TCBs); e.g., anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 TDBs or TCBs; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab), and uses thereof, e.g., for the treatment of cell proliferative disorders (e.g., cancer, e.g., hematological cancers). The pharmaceutical compositions of the present disclosure can be formulated to support relatively low concentrations of a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody or a T cell-engaging bispecific antibody; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab).

[0175] Any of the therapeutic proteins described herein (e.g., an anti-CD3 bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) can be used in the pharmaceutical compositions, although it is understood that other therapeutic proteins can also be used.

[0176] In one aspect, the disclosure provides a pharmaceutical composition comprising a therapeutic protein, a surfactant (e.g., polysorbate 20 (PS20)), a stabilizer (e.g., methionine), a buffer, and a carrier. In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to therapeutic protein 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, 50 to 100, 60 to 80, 70 to 80, or 40 to 50. In certain embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to therapeutic protein is 1 to 100. In some embodiments, the molar ratio of surfactant (e.g., PS20) to therapeutic protein is 45 to 100, 45 to 55, 50 to 100, 60 to 90, 70 to 90, 60 to 80, 70 to 80, 65 to 75, or 75 to 85, e.g., about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80. In certain embodiments, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., mosunetuzumab or lunimotamab) is about 71. In another specific embodiment, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., glofitamab) is about 79. In yet another specific embodiment, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., cebostamab) is about 48. In other embodiments, the molar ratio of surfactant (e.g., P188) to therapeutic protein is 5-50, 5-25, 10-15, or 15-20, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In a specific embodiment, the molar ratio of surfactant (e.g., P188) to therapeutic protein is about 14.In another specific embodiment, the molar ratio of surfactant (eg, P188) to therapeutic protein is about 11.5.

[0177] The pharmaceutical composition can have any suitable concentration of a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody or a T cell-engaging bispecific antibody; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab). For example, in any of the aforementioned pharmaceutical compositions, the concentration of the therapeutic protein can be about 0.01 mg / ml to about 30 mg / ml, e.g., about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, It can be about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 13.5 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, or about 30 mg / ml.

[0178] For example, in some embodiments, the therapeutic protein is anti-CD20 / anti-CD3 TDB, and the concentration of anti-CD20 / anti-CD3 TDB is about 0.1 mg / ml to about 10 mg / ml, about 0.1 mg / ml to about 5 mg / ml, about 0.1 mg / ml to about 3 mg / ml, about 0.1 mg / ml to about 2 mg / ml, about 0.1 mg / ml to about 1.5 mg / ml, about 0.3 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 5 mg / ml, or about 0.5 mg / ml. ml~about 2mg / ml, about 0.6mg / ml~about 2mg / ml, about 0.7mg / ml~about 2mg / ml, about 0.8mg / ml~about 2mg / ml, about 0.9mg / ml~about 10mg / ml, about 0.5mg / ml to about 1.4mg / ml, about 0.5mg / ml to about 1.3mg / ml, about 0.5mg / ml to about 1.2mg / ml, about 0.5mg / ml to about 1.1mg / ml ml, about 0.5 mg / ml to about 1.5 mg / ml, about 0.8 mg / ml to about 1.2 mg / ml, about 1 mg / ml to about 10 mg / ml, about 2 mg / ml to about 10 mg / ml, about 3 mg / ml to about 10 mg / ml, about 4 mg / ml to about 10 mg / ml, about 5 mg / ml to about 10 mg / ml, about 6 mg / ml to about 10 mg / ml, about 7 mg / ml to about 10 mg / ml, about 8 mg / ml to about 10 mg / ml, about 9 mg / ml to about 10 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.8 mg / ml to about 2.2 mg / ml, about 2 mg / ml to about 5 mg / ml, about 3 mg / ml to about 5 mg / ml, or about 4 mg / ml to about 5 mg / ml. In certain embodiments, the concentration of anti-CD20 / anti-CD3 TDB is about 1 mg / ml. In certain embodiments, the concentration of anti-CD20 / anti-CD3 TDB is about 2 mg / ml.

[0179] In some embodiments, the therapeutic protein is anti-HER2 / anti-CD3 TDB, and the concentration of anti-HER2 / anti-CD3 TDB is about 0.1 mg / ml to about 10 mg / ml, about 0.1 mg / ml to about 5 mg / ml, about 0.1 mg / ml to about 3 mg / ml, about 0.1 mg / ml to about 2 mg / ml, about 0.1 mg / ml to about 1.5 mg / ml, about 0.3 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.6 mg / ml to about 2 mg / ml, about 0.7 mg / ml to about 2 mg / ml, about 0.8 mg / ml to about 2 mg / ml, about 0.9 mg / ml to about 10 mg / ml, The concentration of anti-HER2 / anti-CD3 TDB is about 0.5 mg / ml to about 1.4 mg / ml, about 0.5 mg / ml to about 1.3 mg / ml, about 0.5 mg / ml to about 1.2 mg / ml, about 0.5 mg / ml to about 1.1 mg / ml, about 0.5 mg / ml to about 1.5 mg / ml, about 0.8 mg / ml to about 1.2 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.8 mg / ml to about 2.2 mg / ml, about 2 mg / ml to about 5 mg / ml, about 3 mg / ml to about 5 mg / ml, or about 4 mg / ml to about 5 mg / ml. In some embodiments, the concentration of anti-HER2 / anti-CD3 TDB is about 0.5 mg / ml to about 1.5 mg / ml. In a specific embodiment, the concentration of anti-HER2 / anti-CD3 TDB is about 1 mg / ml. In another specific embodiment, the concentration of anti-HER2 / anti-CD3 TDB is about 2 mg / ml.

[0180] In some embodiments, the therapeutic protein is anti-FcRH5 / anti-CD3 TDB, and the concentration of anti-FcRH5 / anti-CD3 TDB is about 0.1 mg / ml to about 10 mg / ml, about 0.1 mg / ml to about 5 mg / ml, about 0.1 mg / ml to about 3 mg / ml, about 0.1 mg / ml to about 2 mg / ml, about 0.1 mg / ml to about 1.5 mg / ml, about 0.3 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.6 mg / ml to about 2 mg / ml, about 0.7 mg / ml to about 2 mg / ml, about 0.8 mg / ml to about 2 mg / ml, about 0.9 mg / ml to about 10 mg / ml, In some embodiments, the concentration of anti-FcRH5 / anti-CD3 TDB is about 0.5 mg / ml to about 1.4 mg / ml, about 0.5 mg / ml to about 1.3 mg / ml, about 0.5 mg / ml to about 1.2 mg / ml, about 0.5 mg / ml to about 1.1 mg / ml, about 0.5 mg / ml to about 1.5 mg / ml, about 0.8 mg / ml to about 1.2 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.8 mg / ml to about 2.2 mg / ml, about 2 mg / ml to about 5 mg / ml, about 3 mg / ml to about 5 mg / ml, or about 4 mg / ml to about 5 mg / ml. In some embodiments, the concentration of anti-FcRH5 / anti-CD3 TDB is about 0.5 mg / ml to about 3 mg / ml. In certain embodiments, the concentration of anti-FcRH5 / anti-CD3 TDB is about 1 mg / ml. In certain embodiments, the concentration of anti-FcRH5 / anti-CD3 TDB is about 1.5 mg / ml. In certain embodiments, the concentration of anti-FcRH5 / anti-CD3 TDB is about 3 mg / ml.

[0181] In some embodiments, the therapeutic protein is anti-CD20 / anti-CD3 TCB, and the concentration of the anti-CD20 / anti-CD3 TCB is about 0.1 mg / ml to about 10 mg / ml, about 0.1 mg / ml to about 5 mg / ml, about 0.1 mg / ml to about 3 mg / ml, about 0.1 mg / ml to about 2 mg / ml, about 0.1 mg / ml to about 1.5 mg / ml, about 0.3 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.5 mg / ml to about 5 mg / ml, about 0.5 mg / ml to about 2 mg / ml, about 0.6 mg / ml to about 2 mg / ml, about 0.7 mg / ml to about 2 mg / ml, about 0.8 mg / ml to about 2 mg / ml, about 0.9 mg / ml to about 10 mg / ml, In some embodiments, the concentration of anti-CD20 / anti-CD3 TCB is about 0.5 mg / ml to about 1.4 mg / ml, about 0.5 mg / ml to about 1.3 mg / ml, about 0.5 mg / ml to about 1.2 mg / ml, about 0.5 mg / ml to about 1.1 mg / ml, about 0.5 mg / ml to about 1.5 mg / ml, about 0.8 mg / ml to about 1.2 mg / ml, about 1 mg / ml to about 5 mg / ml, about 1 mg / ml to about 3 mg / ml, about 1.5 mg / ml to about 2.5 mg / ml, about 1.8 mg / ml to about 2.2 mg / ml, about 2 mg / ml to about 5 mg / ml, about 3 mg / ml to about 5 mg / ml, or about 4 mg / ml to about 5 mg / ml. In some embodiments, the concentration of anti-CD20 / anti-CD3 TCB is about 0.5 mg / ml to about 1.5 mg / ml. In a specific embodiment, the concentration of the anti-CD20 / anti-CD3 TCB is about 1 mg / ml.

[0182] The disclosed pharmaceutical composition comprises a surfactant. Any suitable surfactant can be used. In some embodiments, the surfactant is 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., Super Refined™ 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., Super Refined™ 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)).

[0183] Any suitable concentration of surfactant may be used. The concentration of surfactant in the pharmaceutical composition can be selected based on the desired ratio of surfactant to therapeutic protein and the concentration of the therapeutic protein. 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 0.12% (w / v). In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is about 0.05% (w / v) to about 0.12% (w / v). In a specific embodiment, the surfactant is PS20 and the concentration of PS20 is about 0.06% (w / v). In another specific embodiment, the surfactant is PS20 and the concentration of PS20 is about 0.0.05% (w / v). In yet another specific embodiment, the surfactant is PS20 and the concentration of PS20 is about 0.12% (w / v). In a specific embodiment, the surfactant is P188 and the concentration of P188 is about 0.08% (w / v).

[0184] 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.

[0185] 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.

[0186] 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.

[0187] In pharmaceutical compositions in which the therapeutic protein comprises an Fc region (e.g., an antibody, e.g., a bispecific antibody, e.g., TDB, e.g., an anti-CD20 / anti-CD3 antibody, e.g., mosunetuzumab), the oxidation of methionine at position 257 of the Fc region is less than 10% over two weeks at 40° C. (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% over two weeks at 40° C.). In certain embodiments, the oxidation of methionine at position 257 of the Fc region is about 6% or less over two weeks at 40° C.

[0188] 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 monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, or a mixture thereof.

[0189] In certain examples, the buffer (e.g., histidine, e.g., histidine acetate) is at a concentration of 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, or 9 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 particular 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 some embodiments, the concentration of the buffering agent (e.g., histidine, e.g., histidine acetate or histidine HCl, e.g., L-histidine acetate or L-histidine HCl) is about 10 mM or 20 mM. In particular embodiments, the buffering agent is histidine HCl at a concentration of about 20 mM. In another particular embodiment, the buffering agent is histidine acetate at a concentration of about 20 mM. In yet another particular embodiment, the buffering agent is histidine acetate at a concentration of about 10 mM.

[0190] 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 tonicity agent (e.g., a sugar, such as sucrose) can be at a concentration of about 100 mM to about 500 mM. For example, the tonicity 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, 420 mM to 440 mM, 4 ... The concentration of the tonicity agent can be 0 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 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.

[0191] In some embodiments, the pharmaceutical composition further comprises an antioxidant. In some embodiments, the antioxidant is N-acetyl-DL-tryptophan. In some embodiments, the concentration of N-acetyl-DL-tryptophan is 0.1 mM to 0.5 mM (e.g., about 0.1 mM, about 0.15 mM, about 0.2 mM, about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.45 mM, or about 0.5 mM). In some embodiments, the concentration of N-acetyl-DL-tryptophan is about 0.3 mM.

[0192] 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 5.1 to 6.1. In certain embodiments, the pharmaceutical composition has a pH of about 5.8. In certain embodiments, the pharmaceutical composition has a pH of about 5.5. In certain embodiments, the pharmaceutical composition has a pH of about 5.5. In certain embodiments, the pharmaceutical composition has a pH of about 5.5.

[0193] In addition to the specific ingredients described above, pharmaceutical compositions of the present disclosure can also be prepared by mixing a therapeutic protein having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980)) in the form of a lyophilized formulation 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 or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; carbohydrates such as monosaccharides (e.g., glucose, mannose), disaccharides (e.g., sucrose, trehalose), or polysaccharides (e.g., dextrin), or sugar alcohols, e.g., 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 dispersing agents, e.g., soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., 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).

[0194] 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.

[0195] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., 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 some embodiments, the volume of the liquid formulation for dilution is 10 to 20 ml (e.g., 10 to 15 ml, 15 to 20 ml, 13 to 20 ml, 10 to 17 ml, 13 to 17 ml, or 14 to 16 ml; e.g., 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, or about 20 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 15 ml.

[0196] In another specific embodiment, the liquid formulation for dilution is provided in a container having a volume of about 1 ml or about 2 ml (e.g., about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1 ml, about 1.5 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 liquid formulation for dilution is provided in a container having a volume of about 2.5 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 0.5 ml, about 0.9 ml, or about 1 ml.

[0197] In yet another specific embodiment, the liquid formulation for dilution is provided in a container having a volume of about 15 ml (e.g., 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, or about 20 ml). In some embodiments, the liquid formulation for dilution is provided in a container having a volume of about 10 ml. In some embodiments, the volume of the liquid formulation for dilution is 4 to 12 ml (e.g., 4 to 8 ml, 8 to 12 ml, 4 to 10 ml, 6 to 12 ml, 6 to 10 ml, or 7 to 9 ml; e.g., about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, or 12 ml). In certain embodiments, the volume of the liquid formulation for dilution is about 8 ml.

[0198] 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.

[0199] In some embodiments, after dilution with saline, the concentration of a therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is about 0.001 mg / ml to about 0.6 mg / ml (e.g., about 0.001 mg / ml, about 0.002 mg / ml, about 0.003 mg / ml, about 0.004 mg / ml, about 0.005 mg / ml, 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, or about 0.85 mg / ml). 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, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, or about 0.6 mg / ml). In certain embodiments, after dilution with saline, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., TDB or TCB; e.g., mosunetuzumab, glofitamab, cebostamab, or lunimotamab) is about 0.003 mg / ml, about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.12 mg / ml, about 0.24 mg / ml, or about 0.3 mg / ml. In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TDB; e.g., 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.In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TCB; e.g., glofitamab) is about 0.1 mg / ml or about 0.6 mg / ml. In certain embodiments, after dilution with saline, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-FcRH5 / anti-CD3 bispecific antibody; anti-FcRH5 / anti-CD3 TDB; e.g., cebostamab) is about 0.003 mg / ml, 0.03 mg / ml, or 0.3 mg / ml.

[0200] The formulations herein may also contain two or more active ingredients as needed for the specific indications to be treated, preferably those with complementary activities that do not adversely affect each other.For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or antihormonal agents, such as those mentioned hereinabove).Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0201] 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).

[0202] 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.

[0203] Formulations to be used for in vivo administration are generally sterile, which may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0204] Any pharmaceutical composition described herein may have a shelf life of at least about 12 months (e.g., at least about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 42 months, about 48 months, about 54 months, about 60 months, about 66 months, or 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.

[0205] 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.

[0206] 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.

[0207] 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 by, for example, 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 5° C. for about 36 months or more as assessed by SE-HPLC (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 at about 36 months or more as assessed by SE-HPLC, e.g., 85% to 90%, 90% to 95%, or 95% to 100% at about 5° C. for about 36 months or more as assessed by SE-HPLC. In particular examples, the pharmaceutical composition has a purity of about 95% or greater 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 more at about 5° C.

[0208] 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.).

[0209] III. Exemplary Therapeutic Proteins Therapeutic proteins in the pharmaceutical compositions of the present disclosure include antibodies such as bispecific antibodies, e.g., T cell-dependent bispecific antibodies (TDBs) or T cell-engaging bispecific antibodies (TCBs). The antibodies or bispecific antibodies can bind to CD3 (e.g., anti-CD3 antibodies or anti-CD3 bispecific antibodies). The TDBs can have an anti-CD3 arm having a binding domain that binds to CD3 and an anti-target arm that binds to a target antigen. The anti-target arm of such TDBs can be, for example, an anti-CD20 arm, an anti-FcRH5 arm, or an anti-HER2 arm. The TCBs can have at least one CD3-binding moiety and at least one target antigen-binding moiety. In some embodiments, the TCBs have one CD3-binding moiety and two target antigen-binding moieties. The target antigen-binding moiety of the TCB can be, for example, CD20.

[0210] Anti-CD3 bispecific antibody Anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-CD3 bispecific antibodies (e.g., TDB) described in PCT Publication No. 2015 / 095392, which is incorporated herein by reference in its entirety. In some embodiments, the TDB has an anti-CD3 arm. In some embodiments, the anti-CD3 arm comprises a CD3-binding domain having one or more (e.g., one, two, three, four, five, or all six) hypervariable region (HVR) amino acid sequences of 40G5c. For example, in some embodiments, the CD3-binding domain has HVR-H1 comprising the amino acid sequence of SEQ ID NO:9; HVR-H2 comprising the amino acid sequence of SEQ ID NO:10; HVR-H3 comprising the amino acid sequence of SEQ ID NO:11; HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; HVR-L2 comprising the amino acid sequence of SEQ ID NO:13; and HVR-L3 comprising the amino acid sequence of SEQ ID NO:14.

[0211] In some embodiments, the anti-CD3 antibody comprises 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-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 29-32, respectively. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 15; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 16; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 15, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 16. In certain instances, the CD3 binding domain can have the amino acid sequence of 40G5c, or a derivative or clone thereof. The structural and functional properties of 40G5c are described, for example, in PCT Publication No. WO2015 / 095392, which is incorporated herein by reference in its entirety.

[0212] In other embodiments, the anti-CD3 arm comprises a CD3-binding domain having one or more (e.g., one, two, three, four, five, or all six) hypervariable region (HVR) amino acid sequences of 38E4v1. For example, in some embodiments, the CD3-binding domain has HVR-H1 comprising the amino acid sequence of SEQ ID NO:65; HVR-H2 comprising the amino acid sequence of SEQ ID NO:66; HVR-H3 comprising the amino acid sequence of SEQ ID NO:67; HVR-L1 comprising the amino acid sequence of SEQ ID NO:68; HVR-L2 comprising the amino acid sequence of SEQ ID NO:69; and HVR-L3 comprising the amino acid sequence of SEQ ID NO:70.

[0213] In some embodiments, the anti-CD3 antibody comprises 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: 81-84, 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: 85-88, respectively. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 71; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 72; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 71, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 72. In certain instances, the CD3 binding domain can have the amino acid sequence of 38E4v1, or a derivative or clone thereof. The structural and functional properties of 38E4v1 are described, for example, in PCT Publication No. WO2015 / 095392, which is incorporated herein by reference in its entirety.

[0214] Anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-CD3 T cell activating bispecific antigen binding molecules (e.g., TCBs, e.g., 2+1 TCBs) described in U.S. Patent No. 9,914,776, the entire contents of which are incorporated herein by reference. In some embodiments, the anti-CD3 antibody comprises a CD3-binding domain having one or more (e.g., one, two, three, four, five, or all six) of the following hypervariable regions (HVRs): HVR-H1 comprising the amino acid sequence of SEQ ID NO: 45; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 48; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 49; HVR-L3 comprising the amino acid sequence of SEQ ID NO: 50. For example, in some embodiments, the CD3 binding domain has HVR-H1 comprising the amino acid sequence of SEQ ID NO: 45; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 48; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the CD3-binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 51; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity (e.g., at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 52; or (c) a VH domain similar to (a) and a VL domain similar to (b). In a specific embodiment, the VH domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 51, and the VL domain of the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 52.

[0215] In certain embodiments, the CD3 binding domain binds to an epitope within a fragment of CD3 (eg, human CD3ε) consisting of amino acids 1-26 or 1-27 of human CD3ε.

[0216] The CD3-binding domain of any of the above-described antibodies (e.g., TDB or TCB) can bind to a specific CD3 epitope. For example, the CD3-binding domain can contact amino acids of human CD3ε at a distance of about 3.5 angstroms, about 3.25 angstroms, about 3.0 angstroms, about 2.75 angstroms, or less. In a specific embodiment, the antibody binds to an epitope consisting of 1, 2, 3, 4, or 5 amino acids of human CD3ε at a distance of about 3.5 angstroms, about 3.25 angstroms, about 3.0 angstroms, about 2.75 angstroms, or less. In one embodiment, the antibody contacts amino acids of human CD3ε at a distance of about 3.5 angstroms or less. In a specific embodiment, the antibody binds to an epitope consisting of 1, 2, 3, 4, or 5 amino acids of human CD3ε at a distance of about 3.5 angstroms or less. For example, in certain embodiments, the antibody binds to an epitope of human CD3ε consisting of amino acids selected from Gln1, Asp2, Asn4, Glu6, and Met7. In one particular embodiment, the CD3 binding domain specifically binds to an epitope comprising Glu6. In certain other embodiments, the antibody does not bind to an epitope comprising human CD3ε amino acid Glu5. In certain other embodiments, the antibody does not bind to an epitope comprising human CD3ε amino acids Gly3 and Glu5.

[0217] An anti-CD3 epitope can be determined by an anti-CD3 antibody binding to a peptide fragment of the epitope. Alternatively, the anti-CD3 epitope can be determined by alanine scanning mutagenesis. In certain examples, a reduction of about 20%, about 30%, about 50%, about 80% or more in binding of the anti-CD3 antibody to the mutated CD3 indicates that the amino acid residue of CD3 mutated in the alanine scanning mutagenesis assay is the epitope residue of the anti-CD3 antibody. Alternatively, the anti-CD3 epitope can be determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography.

[0218] In some embodiments, the crystallographically determined epitope is determined using amino acids Q1-M7 of CD3.

[0219] In some embodiments, epitopes determined by crystallography can be determined by first screening a sparse matrix of precipitates in a sitting-drop vapor diffusion format by mixing anti-CD3 antibody Fab dissolved at 10 mg / ml in 0.15 M NaCl, 25 mM Tris, pH 7.5 with a two-fold molar excess (1 mg) of CD3ε peptide. Optimized crystals can be grown from a 1:1 mixture of a reservoir solution containing 70% v / v methyl-pentanediol and 0.1 M HEPES buffer at pH 7.5. The reservoir can be used as a cryoprotectant. Crystals can also be cryogenically transferred by rapid immersion in liquid nitrogen. Diffraction data from the crystals can be collected at the Advanced Photon Source beamline 22ID using a MAR300 CCD detector (Argonne National Laboratory; Lemont, IL). The recorded diffraction data can be integrated and scaled using the program HKL2000 (Z. Otwinowski and W. Minor, "Processing of X-ray Diffraction Data Collected in Oscillation Mode," Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A, pp. 307-326, 1997, C.W. Carter, Jr. & R.M. Sweet, Eds., Academic Press, New York).

[0220] The structures can be phased by molecular replacement (MR) methods using the program Phaser (McCoy, A., et al. Phaser crystallographic software. J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674). For example, the MR search model is the Fab subunit derived from the crystal structure of the HGFA / Fab complex (Protein Data Bank (PDB; Berman HM, Westbrook J., Feng Z., Gilliland G., Bhat TN, Weissig H., Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235-242) code: 2R0L (Wu Y., Eigenbrot C., Liang W.-C., Stawicki S., Shia S., Fan B., Ganesan R., Lipari MT, Kirchhofer D. (2007) Structural insight into distinct mechanisms of protease inhibition by antibodies. Proc. Natl. Acad. Sci. USA 104, 19784-19789). The CD3ε peptide is incorporated into the structure based on the Fo-Fc map.Subsequently, to achieve convergence, the maximum likelihood target function, anisotropic individual B-factor refinement, and TLS (Translation-Libration-Screw-Rotation) refinement methods were used to refine the REFMAC5 (Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr D 53:240-255) and PHENIX (D. Liebschner, P. V. Afonine, M. L. Baker, G. Bunkoczi, V. B. Chen, T. I. Roll, B. Hintze, L.-W. Hung, S. Jain, A. J. McCoy, N. W. Moriarty, R. D. Offner, B. K. Poon, M. G. Prisant, R. J. Read, J. S. Richardson, D. C. Richardson, M. D. Sammito, O. V. Sobolev, D. H. Stockwell, T. C. Terwilliger, A. G. Urzhumtsev, L. L. V. Ideaau, C. J. Williams, and P. D. Adams. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Cryst. (2019). D75, 861-877) may be used to refine the structure.

[0221] In certain other embodiments, the pharmaceutical composition comprises an antibody that comprises a paratope that binds to the same epitope as the anti-CD3 antibody described above.

[0222] Anti-CD20 / anti-CD3 bispecific antibody In embodiments of the present disclosure in which the therapeutic protein is an anti-CD20 / anti-CD3 bispecific antibody, the anti-CD20 / anti-CD3 bispecific antibody can incorporate any of the features, alone or in combination, as described herein.

[0223] Mosunetuzumab In some embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TDB; e.g., mosunetuzumab) comprises an anti-CD20 arm and an anti-CD3 arm. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from: (a) 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); and (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) an HVR-H1 comprising the amino acid sequence of NYYIH (SEQ ID NO: 9); (b) an HVR-H2 comprising the amino acid sequence of WIYPGDGNTKYNEKFKG (SEQ ID NO: 10); (c) an HVR-H3 comprising the amino acid sequence of DSYSNYYFDY (SEQ ID NO: 11); (d) an HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO: 12); (e) an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 13); and (f) an 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-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 29-32, respectively. In some examples, mosunetuzumab is an anti-CD31 antibody comprising: (1) a VH domain comprising: (a) 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-CD31 antibody comprising a first binding domain comprising a VH domain similar to (a) and a VL domain similar to (b). 20 arm, 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 similar to (a) and a VL domain similar to (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.

[0224] 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.

[0225] In some examples, mosunetuzumab is an anti-CD2 antibody comprising: (1) (a) a heavy chain comprising an amino acid sequence of 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 an amino acid sequence of 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; and (c) an anti-CD2 antibody comprising a first binding domain comprising a heavy chain as in (a) and a light chain as in (b). 0 arm, and (2) (a) a heavy chain comprising SEQ ID NO: 35, 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 light chain comprising SEQ ID NO: 36, 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-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.

[0226] The amino acid sequence of mosunetuzumab is summarized in Table 1 below. [Table 1]

[0227] Mosunetuzumab can be produced using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567.

[0228] glofitamab In some other embodiments, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20, comprising a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of YSWIN (SEQ ID NO: 37); HVR-H2 comprising the amino acid sequence of RIFPGDGDTDYNGKFKG (SEQ ID NO: 38); and HVR-H3 comprising the amino acid sequence of NVFDGYWLVY (SEQ ID NO: 39); and a light chain variable region comprising HVR-L1 comprising the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 40); HVR-L2 comprising the amino acid sequence of QMSNLVS (SEQ ID NO: 41); and HVR-L3 comprising the amino acid sequence of AQNLELPYT (SEQ ID NO: 42). and at least one antigen-binding domain that specifically binds to CD3, comprising a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of TYAMN (SEQ ID NO: 45); HVR-H2 comprising the amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO: 46); and HVR-H3 comprising the amino acid sequence of HGNFGNSYVSWFAY (SEQ ID NO: 47); and a light chain variable region comprising HVR-L1 comprising the amino acid sequence of GSSTGAVTTSNYAN (SEQ ID NO: 48); HVR-L2 comprising the amino acid sequence of GTNKRAP (SEQ ID NO: 49); and HVR-L3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 50).

[0229] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises a VH domain having, or comprising, 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 SEQ ID NO: 43, and a VL domain having, or comprising 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 SEQ ID NO: 44, which specifically binds to at least one antigen. and at least one antigen-binding domain that specifically binds to CD3, comprising 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 SEQ ID NO: 51, or a VH domain comprising that sequence, and a VL domain having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) to SEQ ID NO: 52, or a VL domain comprising that sequence.

[0230] In one embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is an antibody fragment, particularly a Fab molecule or an scFv molecule, more particularly a Fab molecule. In a specific embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced with each other).

[0231] In one embodiment, the antigen-binding domain that specifically binds to CD20 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is an antibody fragment, particularly a Fab or scFv molecule, more particularly a Fab molecule. In a specific embodiment, the antigen-binding domain that specifically binds to CD20 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) is a conventional Fab molecule.

[0232] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises at least one antigen-binding domain that specifically binds to CD20 and at least one antigen-binding domain that specifically binds to CD3. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises a first antigen-binding domain that specifically binds to CD3 and a second and third antigen-binding domain that specifically bind to CD20. In one embodiment, the first antigen-binding domain is a crossover Fab molecule, and the second and third antigen-binding domains are each conventional Fab molecules. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) further comprises an Fc domain. Anti-CD20 / anti-CD3 bispecific antibodies may comprise modifications of the Fc region and / or antigen-binding domain as described herein. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce Fc receptor binding and / or effector function. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and P329G (EU numbering).

[0233] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises an antigen-binding domain that specifically binds CD3 fused to the N-terminus of a first subunit of an Fc domain at the C-terminus of a Fab heavy chain; a first antigen-binding domain that specifically binds CD20 fused to the N-terminus of a Fab heavy chain of the antigen-binding domain that specifically binds CD3 at the C-terminus of the Fab heavy chain; and a second antigen-binding domain that specifically binds CD20 fused to the N-terminus of a second subunit of an Fc domain at the C-terminus of the Fab heavy chain.

[0234] In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises: (a) a first Fab molecule that specifically binds to CD3, in particular CD3 epsilon, wherein the variable domains VL and VH of the Fab light and heavy chains are exchanged with each other; and (b) a second and a third Fab molecule that specifically binds to CD20, wherein in the constant domain CL of the second and third Fab molecules, the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by a lysine (K). ) or arginine (R), in particular by arginine (R) (Kabat numbering), and in the constant domain CH1 of the second and third Fab molecules, the amino acid at position 147 is substituted by glutamic acid (E) (EU numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (EU numbering), the second and third Fab molecules; and (c) an Fc domain composed of the first and second subunits capable of stable association.

[0235] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, e.g., glofitamab) comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0236] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (eg, an anti-CD20 / anti-CD3 TCB, eg, glofitamab) is bivalent with respect to CD20 and monovalent with respect to CD3.

[0237] In one embodiment, a first Fab molecule (i.e., binds CD3) is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, a second Fab molecule (i.e., binds CD20) is fused at the C-terminus of its Fab heavy chain to the N-terminus of the heavy chain of the first Fab molecule, and a third Fab molecule (i.e., binds CD20) is fused at the C-terminus of its Fab heavy chain to the N-terminus of the other subunit of the Fc domain. In one embodiment, the first Fab molecule comprises a VH domain at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 51 and a VL domain at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52. In yet a further embodiment, the first Fab molecule comprises a heavy chain variable region sequence comprising the amino acid sequence of SEQ ID NO: 51 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52.

[0238] In one embodiment, the second Fab molecule and the third Fab molecule each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 44.

[0239] In one embodiment, the second and third Fab molecules each comprise the heavy chain variable region sequence of SEQ ID NO:43 and the light chain variable region sequence of SEQ ID NO:44.

[0240] In certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB; e.g., glofitamab) comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 56. In further specific embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 53, the polypeptide sequence of SEQ ID NO: 54, the polypeptide sequence of SEQ ID NO: 55, and the polypeptide sequence of SEQ ID NO: 56. In a more specific embodiment, the bispecific antibody comprises one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 53, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 54, one polypeptide chain comprising the amino acid sequence of SEQ ID NO: 55, and two polypeptide chains each comprising the amino acid sequence of SEQ ID NO: 56.

[0241] Certain anti-CD20 / anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 and European Patent Application Nos. EP 15188093 and EP 16169160, each of which is incorporated by reference in its entirety.

[0242] The sequence of glofitamab is summarized in Table 2. [Table 2]

[0243] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the pharmaceutical composition of the present disclosure is glofitamab.

[0244] Anti-FcRH5 / anti-CD3 bispecific antibody In embodiments of the present disclosure in which the therapeutic protein is an anti-FcRH5 / anti-CD30 bispecific antibody, the anti-FcRH5 / anti-CD30 bispecific antibody can incorporate any of the features, alone or in combination, as described herein.

[0245] Sebostamab In some embodiments, an anti-FcRH5 / anti-CD3 bispecific antibody (eg, anti-FcRH5 / anti-CD3 TDB; eg, cebostamab) comprises an anti-FcRH5 arm and an anti-CD3 arm. In some embodiments, the anti-FcRH5 / anti-CD3 bispecific antibody has 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 RFGVH (SEQ ID NO: 57); (b) an HVR-H2 comprising the amino acid sequence of VIWRGGSTDYNAAFVS (SEQ ID NO: 58); (c) an HVR-H3 comprising the amino acid sequence of HYYGSSDYALDN (SEQ ID NO: 59); (d) an HVR-L1 comprising the amino acid sequence of KASQDVRNLVV (SEQ ID NO: 60); (e) an HVR-L2 comprising the amino acid sequence of SGSYRYS (SEQ ID NO: 61); or (f) an HVR-L3 comprising the amino acid sequence of QQHYSPPYT (SEQ ID NO: 62). 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) an HVR-H1 comprising the amino acid sequence of SYYIH (SEQ ID NO: 65); (b) an HVR-H2 comprising the amino acid sequence of WIYPENDNTKYNEKFKD (SEQ ID NO: 66); (c) an HVR-H3 comprising the amino acid sequence of DGYSRYYFDY (SEQ ID NO: 67); (d) an HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO: 68); (e) an HVR-L2 comprising the amino acid sequence of WTSTRKS (SEQ ID NO: 69); and (f) an HVR-L3 comprising the amino acid sequence of KQSFILRT (SEQ ID NO: 70).In some examples, cebostamab comprises: (1) an anti-FcRH5 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: 73-76, 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: 77-80, 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: 81-84, 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: 85-88, respectively. In some examples, cebostamab is an anti-Fc antibody comprising: (1) a VH domain comprising: (a) SEQ ID NO: 63, 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: 64, 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-Fc antibody comprising a first binding domain comprising a VH domain similar to (a) and a VL domain similar to (b). an RH5 arm; and (2) (a) a VH domain comprising SEQ ID NO: 71, 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: 72, 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-CD3 arm comprising a second binding domain comprising a VH domain similar to (a) and a VL domain similar to (b).In some examples, cebostamab comprises (1) an anti-FcRH5 arm comprising a first binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 63 and a VL domain comprising the amino acid sequence of SEQ ID NO: 64, and (2) an anti-CD3 arm comprising a second binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 71 and a VL domain comprising the amino acid sequence of SEQ ID NO: 72.

[0246] In some examples, cebostamab is described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN:List 84, Vol. 34, No. 3, published 2020 (see page 701), has CAS Registry Number 1905409-39-3, and has (1) an anti-FcRH5 arm comprising the heavy and light chain sequences of SEQ ID NOs: 89 and 90, respectively; and (2) an anti-CD3 arm comprising the heavy and light chain sequences of SEQ ID NOs: 91 and 92, respectively.

[0247] In some examples, cebostamab is an anti-FcRH antibody comprising: (1) (a) a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 89, or 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 light chain comprising an amino acid sequence set forth in SEQ ID NO: 90, or having at least 90% sequence identity thereto (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); and (c) an anti-FcRH antibody comprising a first binding domain comprising a heavy chain as in (a) and a light chain as in (b). 5 arms, and (2) (a) a heavy chain comprising SEQ ID NO: 91, 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 light chain comprising SEQ ID NO: 92, 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-CD3 arm comprising a second binding domain comprising a heavy chain as in (a) and a light chain as in (b). In some examples, cebostamab comprises (1) an anti-FcRH5 arm comprising a first binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 89 and a light chain comprising the amino acid sequence of SEQ ID NO: 90, and (2) an anti-CD3 arm comprising a second binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 92.

[0248] The amino acid sequence of cebostamab is summarized in Table 3 below. [Table 3]

[0249] Sebostamab can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567.

[0250] Anti-HER2 / anti-CD3 bispecific antibody In embodiments of the present disclosure in which the therapeutic protein is an anti-HER2 / anti-CD3 bispecific antibody, the anti-HER2 / anti-CD3 bispecific antibody can incorporate any of the features, alone or in combination, as described herein.

[0251] Lunimotamab In some embodiments, an anti-HER2 / anti-CD3 bispecific antibody (e.g., anti-HER2 / anti-CD3 TDB; e.g., lunimotamab) comprises an anti-HER2 arm and an anti-CD3 arm. In some embodiments, the anti-HER2 / anti-CD3 bispecific antibody has a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 comprising the amino acid sequence of DTYIH (SEQ ID NO: 93); (b) an HVR-H2 comprising the amino acid sequence of RIYPTNGYTRYADSVKG (SEQ ID NO: 94); (c) an HVR-H3 comprising the amino acid sequence of WGGDGFYAMDY (SEQ ID NO: 95); (d) an HVR-L1 comprising the amino acid sequence of RASQDVNTAVA (SEQ ID NO: 96); (e) an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 97); and (f) an HVR-L3 comprising the amino acid sequence of QQHYTTPPT (SEQ ID NO: 98). an anti-HER2 arm; 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: 109); (b) HVR-H2 comprising the amino acid sequence of WIYPGDGNTKYNEKFKG (SEQ ID NO: 110); (c) HVR-H3 comprising the amino acid sequence of DSYSNYYFDY (SEQ ID NO: 111); (d) HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO: 112); (e) HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 113); and (f) HVR-L3 comprising the amino acid sequence of TQSFILRT (SEQ ID NO: 114).In some examples, lunimotamab is an anti-H1 antibody comprising: (1) 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: 101-104, 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: 105-108, 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, each comprising the sequences of SEQ ID NOs: 117-120, 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, each comprising the sequences of SEQ ID NOs: 121-124. In some examples, lunimotamab is an anti-HE antibody comprising: (1) a first binding domain comprising: (a) a VH domain comprising SEQ ID NO: 99, 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: 100, 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-HE antibody comprising a first binding domain comprising a VH domain similar to (a) and a VL domain similar to (b). an R2 arm; and (2) (a) a VH domain comprising SEQ ID NO: 115, 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: 116, 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-CD3 arm comprising a second binding domain comprising a VH domain similar to (a) and a VL domain similar to (b).In some examples, lunimotamab comprises (1) an anti-HER2 arm comprising a first binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 99 and a VL domain comprising the amino acid sequence of SEQ ID NO: 100, and (2) an anti-CD3 arm comprising a second binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 115 and a VL domain comprising the amino acid sequence of SEQ ID NO: 116.

[0252] In some examples, lunimotamab has International Nonproprietary Name (INN) List 124 (WHO Drug Information, Vol. 34, No. 4, 2020, p. 1031) or CAS Registry Number 2361325-98-4, and has (1) an anti-HER2 arm comprising the heavy chain and light chain sequences of SEQ ID NOs: 125 and 126, respectively, and (2) an anti-CD3 arm comprising the heavy chain and light chain sequences of SEQ ID NOs: 128 and 127, respectively.

[0253] In some examples, lunimotamab is an anti-HER2 antibody comprising: (1) (a) a heavy chain comprising SEQ ID NO: 125, 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 light chain comprising SEQ ID NO: 126, 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); and (c) an anti-HER2 antibody comprising a first binding domain comprising a heavy chain as in (a) and a light chain as in (b). and (2) (a) a heavy chain comprising SEQ ID NO: 127, 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 light chain comprising SEQ ID NO: 128, 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-CD3 arm comprising a second binding domain comprising a heavy chain as in (a) and a light chain as in (b). In some examples, lunimotamab comprises (1) an anti-HER2 arm comprising a first binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 125 and a light chain comprising the amino acid sequence of SEQ ID NO: 126, and (2) an anti-CD3 arm comprising a second binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 127 and a light chain comprising the amino acid sequence of SEQ ID NO: 128.

[0254] The amino acid sequence of lunimotamab is summarized in Table 4 below. [Table 4]

[0255] Lunimotamab can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567.

[0256] IV. ANTIBODY PROPERTIES, MODIFICATIONS, AND METHODS FOR CHARACTERIZATION The pharmaceutical compositions provided herein may further comprise any of the antibodies described above, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above, wherein one or both of the variable domain sequences comprise a post-translational modification. Additionally or alternatively, an antibody according to any of the above embodiments (e.g., a bispecific antibody, e.g., TDB or TCB) may incorporate any of the features, alone or in combination, as described below.

[0257] 1. Antibody affinity In certain embodiments, the antibodies of the pharmaceutical compositions and methods provided herein have a concentration 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 -13M , e.g. 10 -9 M~10 -13 Dissociation constant (K D )

[0258] In one embodiment, K D is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 The [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation can be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN® 20) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20 (商標) Packard) and the plate is TOPCOUNT (商標) Count for 10 minutes on a gamma counter (Packard). Concentrations of each Fab that yield 20% or less of maximal binding are selected for use in competitive binding assays.

[0259] According to another embodiment, K Dis measured using a BIACORE® surface plasmon resonance assay. For example, assays using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) are performed at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN® 20) surfactant (PBST) are injected at a flow rate of approximately 25 μL / min at 25°C. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). D ) is k off / k on The ratio is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on rate by the surface plasmon resonance assay is 10 6 M- 1 s- 1 If the on rate exceeds , the on rate is measured using a spectrophotometer (Aviv Instruments) with stopped flow or an 8000 series SLM-AMINCO with a stirred cuvette. (商標)It can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations, as measured in a spectrometer such as a spectrophotometer (ThermoSpectronic).

[0260] 2. Antibody fragment In certain embodiments, the therapeutic protein is an antibody fragment, e.g., an antibody fragment that binds to CD3 and CD20. Antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that comprise salvage receptor binding epitope residues and have increased in vivo half-lives.

[0261] Diabody is an antibody fragment that has two antigen binding sites, and can be bivalent or bispecific.See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al. Nat.Med. 9:129-134 (2003); and Hollinger et al. Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993).Triabody and tetrabody are also described in Hudson et al. Nat.Med. 9:129-134 (2003).

[0262] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0263] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0264] 3. Chimeric and humanized antibodies In certain embodiments, the therapeutic protein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0265] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues of a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0266] Humanized antibodies and methods for their production are reviewed by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005)) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498(1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68(2005) and Klimka et al., Br. J. Cancer, 83:252-260(2000) (describing a "guided selection" approach to FR shuffling).

[0267] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0268] 4. Knob-in-hole Antibody Manipulation Antibodies (e.g., bispecific antibodies, such as TDB or TCB) can be prepared as full-length antibodies or antibody fragments. Techniques for generating bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Using "knob-in-hole" engineering of bispecific antibodies, a first subunit of an Fc domain containing a knob (e.g., a subunit of an Fc domain in a first arm) and a second subunit of an Fc domain containing a hole to which the knob of the first arm can bind (e.g., a subunit of an Fc domain in a second arm) can be generated. The knob of a bispecific antibody, in one embodiment, may be on one subunit of the Fc domain (e.g., the Fc subunit of an anti-CD3 arm). Alternatively, the knob of a bispecific antibody of the present disclosure may be on another subunit of the Fc domain (e.g., the anti-target arm; e.g., the Fc subunit of an anti-CD20, anti-FcRH5, or anti-HER2 arm). The hole of a bispecific antibody of the present disclosure, in one embodiment, may be on the anti-CD3 arm. Alternatively, the hole of a bispecific antibody of the present disclosure may be on the anti-target arm (e.g., the anti-CD20, anti-FcRH5, or anti-HER2 arm). In some examples, an anti-CD20 / anti-CD3 bispecific antibody produced using knobs-in-hole technology can comprise one or more heavy chain constant domains, where the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some examples, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain.In some examples, the CH31 domain and the CH32 domain each comprise a protrusion or a cavity, and the protrusion or cavity in the CH31 domain can be located in the cavity or protrusion in the CH32 domain, respectively. In some examples, the CH31 and the CH32 domain meet at the interface between the protrusion and the cavity. In some examples, the CH21 domain and the CH22 domain each comprise a protrusion or a cavity, and the protrusion or cavity in the CH21 domain can be located in the cavity or protrusion in the CH22 domain, respectively. In some examples, the CH21 and the CH22 domain contact at the interface between the protrusion and the cavity.

[0269] Bispecific antibodies can also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Bispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994)); and, for example, by the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0270] Bispecific antibodies or antibody fragments thereof may also include "dual-acting Fabs" or "DAFs" that contain antigen-binding sites that bind to target antigens such as CD3 and CD20, FcRH5, or HER2 (e.g., U.S. Patent Application Publication No. 2008 / 0069820; Bostrom et al., 2009, Science 323:1610-14).

[0271] 5. Antibody Variants In some instances, amino acid sequence variants of the above-described antibodies are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct retains the desired characteristics, e.g., antigen binding.

[0272] (i) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and framework regions (FRs). Conservative substitutions are shown in Table 5 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 5 under the heading of "Exemplary Substitutions," and are as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 5]

[0273] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0274] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0275] One type of substitutional variant involves one or more hypervariable region (HVR) residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0276] Alterations (e.g., substitutions) may be made, for example, in HVRs to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or antigen-facing residues, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from a secondary library is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0277] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within HVRs. Such changes may, for example, be outside the antigen contact residues within the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0278] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they possess desired properties.

[0279] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0280] (ii) glycosylation variants In some examples, the antibodies of the pharmaceutical compositions provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0281] If the antibody contains an Fc region, the carbohydrate attached thereto may vary. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are commonly attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications in the antibodies of the present disclosure may be performed to generate antibody variants with improved specific properties.

[0282] In some examples, the antibody of the pharmaceutical composition has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total amount of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures) measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located approximately at position 297 (EU numbering of Fc region residues) in the Fc region; however, due to slight sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; 0132140; 2004 / 0110704; 2004 / 0110282; 2004 / 0109865; WO 2003 / 085119; 2003 / 084570; 2005 / 035586; 2005 / 035778; 2005 / 053742; 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108, Presta, L; and International Publication No. WO2004 / 056312, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. WO2003 / 085107).

[0283] In view of the above, in some examples, antibodies useful in the present disclosure comprise an aglycosylation site mutation. In some examples, the aglycosylation site mutation reduces the effector function of the bispecific antibody. In some examples, the aglycosylation site mutation is a substitution mutation. In some examples, the bispecific antibody comprises a substitution mutation in the Fc region that reduces the effector function. In some examples, the substitution mutation is at amino acid residues N297, L234, L235, D265, and / or P329 (EU numbering). In some examples, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some examples, the substitution mutation is at amino acid residue N297. In a preferred embodiment, the substitution mutation is N297A.

[0284] In another example, antibody variants containing bisected oligosaccharides are used according to the methods of the present disclosure, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0285] (iii) Fc region variant In some examples, antibody variants have one or more amino acid modifications introduced into the Fc region (i.e., Fc region variants (see, e.g., US2012 / 0251531)) of an antibody (e.g., a bispecific antibody, e.g., TDB or TCB) and can be administered to a subject with cancer (e.g., a B-cell proliferative disorder) according to the methods of the present disclosure. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0286] In some instances, bispecific Fc region antibody variants retain some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9, 457-492 (1991), p. 464, Table 3. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc., Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (PROMEGA®, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo in an animal model such as that disclosed in Clynes et al. Proc. Natl Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind to C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. Immunol. 18(12):1759-1769 (2006)).

[0287] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0288] In a particular example, the proline at position 329 of the wild-type human Fc region in the antibody is substituted with an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of FcγRIII (Sondermann et al. Nature. 406, 267-273, 2000), or with glycine or arginine. In certain embodiments, the bispecific antibody has at least one additional amino acid substitution. In one embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S; in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region (see, e.g., US 2012 / 0251531); and in yet another embodiment, the at least one additional amino acid substitution is L234A, L235A, and P329G in the human IgG1 Fc region.

[0289] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0290] In certain cases, the therapeutic protein is an antibody having an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0291] In some examples, changes are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0292] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0293] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0294] (iv) Cysteine-engineered antibody variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of a bispecific antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are positioned at accessible sites of the bispecific antibody, which can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0295] The present invention also specifically contemplates immunoconjugates of antibodies conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0296] In some examples, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the bispecific antibody is selected from the group consisting of maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,...

Claims

1. 1. A pharmaceutical composition comprising a therapeutic protein, polysorbate 20 (PS20), methionine, histidine, sucrose, and water, wherein a molar ratio of the PS20 to the therapeutic protein is less than or equal to 80, the therapeutic protein is at a concentration of about 3 mg / ml or about 1 mg / ml, the PS20 is at a concentration of 0.05% to 0.06% weight / volume (w / v), the methionine is at a concentration of about 10 mM, the histidine is at a concentration of about 10 mg / ml or about 20 mg / ml, the sucrose is at a concentration of about 240 mg / ml, and the pH of the pharmaceutical composition is 5.5 to 6.1; The therapeutic protein is (a) a T cell-dependent bispecific antibody (TDB) comprising an anti-CD3 arm comprising a CD3 binding domain, an anti-target arm comprising a target binding domain, and an Fc domain comprising a first subunit and a second subunit, wherein the CD3 binding domain is a Fab and the target binding domain is a Fab; or (b) a T cell engaging bispecific antibody (TCB) comprising a CD3 binding domain comprising a crossover Fab molecule, a first target binding domain comprising a first Fab molecule, a second target binding domain comprising a second Fab molecule, and an Fc domain comprising a first subunit and a second subunit, wherein the CD3 binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit, the first target binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the CD3 binding domain, and the second target binding domain is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit.

23. The pharmaceutical composition according to claim 22,

2. 10. The pharmaceutical composition of claim 1, formulated as a drug product (DP).

3. 2. The pharmaceutical composition of claim 1, wherein the histidine is histidine acetate or histidine HCl.

4. 10. The pharmaceutical composition of claim 1, further comprising an antioxidant.

5. 5. The pharmaceutical composition of claim 4, wherein the antioxidant is N-acetyl-DL-tryptophan.

6. 6. The pharmaceutical composition of claim 5, wherein the concentration of N-acetyl-DL-tryptophan is 0.1 to 0.5 mM.

7. 7. The pharmaceutical composition of claim 6, wherein the concentration of N-acetyl-DL-tryptophan is about 0.3 mM.

8. 2. The pharmaceutical composition of claim 1, wherein the bispecific antibody has a methionine at position 257 (EU numbering) of the Fc region, and the oxidation of the methionine at position 257 of the Fc region is less than about 10% over two weeks at 40° C.

9. 9. The pharmaceutical composition of claim 8, wherein the oxidation of methionine at position 257 of the Fc region is less than or equal to about 6% over a period of two weeks at 40°C.

10. The pharmaceutical composition of claim 1, wherein the therapeutic protein is TDB and the anti-target arm is an anti-FcRH5 arm.

11. the anti-CD3 arm (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:65; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 66; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:68; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and (vi) a CD3-binding domain comprising HVR-L3 comprising the amino acid sequence of SEQ ID NO:

70.

12. 12. The pharmaceutical composition of claim 11, wherein the CD3 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 71; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; or (c) a VH domain similar to (a) and a VL domain similar to (b).

13. The pharmaceutical composition of claim 12, wherein the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 71 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:

72.

14. the anti-FcRH5 arm (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:57; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:58; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:59; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 61; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 62;

15. 15. The pharmaceutical composition of claim 14, wherein the FcRH5 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 63; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 64; or (c) a VH domain similar to (a) and a VL domain similar to (b).

16. The pharmaceutical composition of claim 15, wherein the VH domain of the FcRH5 binding domain comprises the amino acid sequence of SEQ ID NO: 63 and the VL domain of the FcRH5 binding domain comprises the amino acid sequence of SEQ ID NO:

64.

17. The pharmaceutical composition of claim 1, wherein the therapeutic protein is TDB and the anti-target arm is an anti-HER2 arm.

18. the anti-CD3 arm (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 111; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 112; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 113; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 114 20. The pharmaceutical composition of claim 17, comprising a CD3 binding domain comprising:

19. 19. The pharmaceutical composition of claim 18, wherein the CD3 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 115; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 116; or (c) a VH domain similar to (a) and a VL domain similar to (b).

20. The pharmaceutical composition of claim 19, wherein the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 115 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:

116.

21. the anti-HER2 arm comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 93; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 94; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 95; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 96; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 97; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 98 20. The pharmaceutical composition of claim 17, comprising a HER2 binding domain comprising:

22. 22. The pharmaceutical composition of claim 21 , wherein the HER2 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 99; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 100; or (c) a VH domain similar to (a) and a VL domain similar to (b).

23. 23. The pharmaceutical composition of claim 22, wherein the VH domain of the HER2 binding domain comprises the amino acid sequence of SEQ ID NO: 99 and the VL domain of the HER2 binding domain comprises the amino acid sequence of SEQ ID NO:

100.

24. The pharmaceutical composition of claim 1, wherein the therapeutic protein is TDB and the anti-target arm is an anti-CD20 arm.

25. The method of claim 20, wherein the anti-CD3 arm is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 25. The pharmaceutical composition of claim 24, comprising a CD3 binding domain comprising:

26. The pharmaceutical composition of claim 25, wherein the CD3 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) a VH domain similar to (a) and a VL domain similar to (b).

27. ​​The pharmaceutical composition described in claim 26, wherein the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 15 and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:

16.

28. The method of claim 1, wherein the anti-CD20 arm is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:6 25. The pharmaceutical composition of claim 24, comprising a CD20 binding domain comprising:

29. The pharmaceutical composition of claim 28, wherein the CD20 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8; or (c) a VH domain similar to (a) and a VL domain similar to (b).

30. The pharmaceutical composition of claim 29, wherein the VH domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO:7, and the VL domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO:

8.

31. The pharmaceutical composition of claim 1, wherein the therapeutic protein is TCB and the target binding domain is a CD20 binding domain.

32. The CD3 binding domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 45; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 48; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 50 32. The pharmaceutical composition of claim 31 , comprising:

33. The pharmaceutical composition of claim 32, wherein the CD3 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:51; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:52; or (c) a VH domain similar to (a) and a VL domain similar to (b).

34. The pharmaceutical composition of claim 33, wherein the VH domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:51, and the VL domain of the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:

52.

35. The CD20 binding domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 42 32. The pharmaceutical composition of claim 31 , comprising:

36. The pharmaceutical composition of claim 35, wherein the CD20 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 43; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44; or (c) a VH domain similar to (a) and a VL domain similar to (b).

37. The pharmaceutical composition of claim 36, wherein the VH domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO: 43, and the VL domain of the CD20 binding domain comprises the amino acid sequence of SEQ ID NO:

44.

38. The pharmaceutical composition of claim 1 , wherein the therapeutic protein is an IgG antibody.

39. The IgG antibody is IgG １ 39. The pharmaceutical composition of claim 38 which is an antibody.

40. 39. The pharmaceutical composition of claim 38, wherein the bispecific antibody comprises one or more substitution mutations in the Fc region.

41. 41. The pharmaceutical composition of claim 40, wherein the bispecific antibody comprises an aglycosylation site mutation.

42. The pharmaceutical composition of claim 41 , wherein the aglycosylation site mutation is a substitution mutation.

43. 41. The pharmaceutical composition of claim 40, wherein the one or more substitution mutations reduce an effector function of the bispecific antibody.

44. 41. The pharmaceutical composition of claim 40, wherein the one or more substitution mutations are at one or more amino acid residues selected from the group consisting of N297, L234, L235, D265 and P329 (EU numbering).

45. 45. The pharmaceutical composition of claim 44, wherein the substitution mutation is at least one selected from the group consisting of N297A, N297G, L234A, L235A, D265A, and P329G (EU numbering).

46. 41. The pharmaceutical composition of claim 40, wherein the one or more substitution mutations in the Fc region comprise one or more knob-in-hole mutations.

47. the first subunit comprises T366W and N297G substitution mutations and the second subunit comprises T366S, L368A, Y407V and N297G substitution mutations (EU numbering); or The second subunit comprises T366W and N297G substitution mutations, and the first subunit comprises T366S, L368A, Y407V and N297G substitution mutations (EU numbering); 2. The pharmaceutical composition of claim 1.

48. 2. The pharmaceutical composition of claim 1, wherein the therapeutic protein is cebostamab.

49. The pharmaceutical composition of claim 1 , wherein the therapeutic protein is lunimotamab.

50. The pharmaceutical composition of claim 1, wherein the CD3 binding domain specifically binds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide.

51. 51. The pharmaceutical composition of claim 50, wherein the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3ε polypeptide or a cyno CD3ε polypeptide, respectively.

52. 51. The pharmaceutical composition of claim 50, wherein the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3γ polypeptide or a cyno CD3γ polypeptide, respectively.

53. The pharmaceutical composition of claim 1 , wherein the therapeutic protein is a monoclonal antibody.

54. The pharmaceutical composition of claim 1 , wherein the therapeutic protein is a human, humanized or chimeric antibody.

55. 55. The pharmaceutical composition of any one of claims 1 to 54, in unit dosage form.

56. 56. The pharmaceutical composition of claim 55, wherein the unit dosage form is a liquid formulation for dilution.

57. 57. The pharmaceutical composition of claim 56, wherein the liquid formulation for dilution is provided in a container having a volume of about 50 ml, about 15 ml, about 2 ml, or about 1 ml.

58. 57. The pharmaceutical composition of claim 56, wherein the volume of the liquid formulation for dilution is about 15 ml, about 30 ml, about 8 ml, about 15 ml, about 0.5 ml, or about 0.9 ml.

59. 57. The pharmaceutical composition of claim 56, wherein the liquid formulation for dilution is for dilution with saline containing 0.45% or 0.9% (w / v) NaCl.

60. (a) containing 1,000 or less particles having a diameter of 2 μm or greater per ml as detected by high accuracy particle counting (HIAC); (b) has a shelf life of at least 36 months when stored at 5° C.±3° C. and protected from light; (c) is stable through one or more freeze-thaw cycles; (d) is stable at about 25° C. for about 2 weeks or more; and / or (e) stable for about 48 months or more at −20° C.; A pharmaceutical composition according to any one of claims 1 to 54.

61. 61. The pharmaceutical composition of claim 60, 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.

62. 62. The pharmaceutical composition of claim 61, which is determined to be stable if it maintains a purity that changes less than 5% as measured by SE-HPLC or by non-reducing CE-SDS assay.

63. 63. The pharmaceutical composition of claim 62, wherein the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

64. (a) the pharmaceutical composition has a purity of about 85% or greater as assessed by SE-HPLC; (b) the purity of said pharmaceutical composition, as assessed by SE-HPLC, remains substantially the same for greater than about 36 months at about 5° C.; (c) the pharmaceutical composition has a purity of about 75% or greater as assessed by a non-reducing CE-SDS assay; and / or (d) the purity of the pharmaceutical composition, as assessed by a non-reducing CE-SDS assay, is maintained for greater than about 36 months at about 5° C.; A pharmaceutical composition according to any one of claims 1 to 54.

65. 65. The pharmaceutical composition of claim 64, wherein the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

66. 55. The pharmaceutical composition of any one of claims 1 to 54, formulated for intravenous administration.

67. 55. The pharmaceutical composition of any one of claims 1 to 54, which does not contain a preservative.

68. 55. The pharmaceutical composition of any one of claims 1 to 54, formulated for administration by injection after dilution with saline containing 0.45% or 0.9% NaCl.

69. A medicament comprising the pharmaceutical composition according to any one of claims 1 to 54, (i) treating or slowing the progression of a cell proliferative disorder in a subject in need thereof; or (ii) enhancing immune function in a subject with a cell proliferative disorder Medicine for.

70. The pharmaceutical composition of claim 69, wherein the cell proliferative disorder is cancer.

71. The pharmaceutical of claim 69, wherein the therapeutic protein is formulated to bind to a CD3 molecule located on an immune effector cell and to a target molecule located on a target cell other than the immune effector cell.

72. The pharmaceutical of claim 71, wherein the bispecific antibody activates the immune effector cell after binding to the CD3 molecule and the target molecule.

73. The pharmaceutical of claim 72, wherein the activated immune effector cells are capable of exerting a cytotoxic and / or apoptotic effect on the target cells.

74. The cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL), 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's lymphoma, B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, intermediate B-cell lymphoma, and endothelial 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, 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 (LPLA), Lymphoma (LL), Waldenström'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, 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 myeloma, primary effusion lymphoma, breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, multiple myeloma, kidney cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia (APL), chronic myeloproliferative disorder, thrombocytopenic leukemia, precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursor T-cell acute lymphoblastic leukemia (pre-T-ALL), mast cell disease, mast cell leukemia, mast cell sarcoma, myeloid sarcoma, lymphocytic leukemia,Or undifferentiated leukemia.

75. The method of claim 69, wherein the therapeutic protein is a bispecific antibody formulated for administration to the subject in a dosage of about 10 μg to about 100 mg.

76. The method of claim 75, wherein the therapeutic protein is a bispecific antibody formulated for administration to the subject in a dosage of about 1 mg to about 60 mg.

77. 70. The method of claim 69, wherein the subject is co-administered with at least one additional therapeutic agent.

78. The pharmaceutical composition of claim 69, wherein the subject is a human.

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