Pharmaceutical compositions of therapeutic proteins and methods of use

Abstract

The present disclosure provides pharmaceutical compositions of therapeutic proteins and methods of use thereof.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on March 22, 2023, is named 50474-276WO2_Sequence_Listing_3_22_23, and is 119,559 bytes in size. Technical Field

[0003] The present disclosure relates to compositions (eg, pharmaceutical compositions) comprising therapeutic proteins (eg, T cell-dependent bispecific antibodies or T cell-engaging bispecific antibodies) and methods of use thereof. Background Art

[0004] 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, cevostamab, or runimotamab) are increasingly used to treat various diseases and conditions, such as cancer and autoimmunity. Such therapeutic proteins can be formulated in aqueous carriers for administration to a subject, e.g., intravenously or subcutaneously. During storage, handling, and administration of such pharmaceutical compositions, it is necessary to reduce the loss of therapeutic proteins, which can occur through degradation and surface adsorption (e.g., adsorption of proteins to surfaces of filters, storage tanks, tubing, syringes, intravenous infusion bags, and other containers). In situations where the pharmaceutical composition contains a relatively low concentration of a therapeutic protein, these factors may significantly increase protein loss, resulting in reduced therapeutic efficacy of the pharmaceutical composition.

[0005] Therefore, there is a need in the art to develop pharmaceutical formulations in which therapeutic proteins (eg, low-dose therapeutic proteins) remain stable and avoid loss due to adsorption. Summary of the Invention

[0006] The present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising therapeutic proteins (e.g., low concentration therapeutic proteins, such as 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., mosulatuzumab, gefuzumab, silvosetumab, or rumatuzumab)) 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 the therapeutic protein with little to no protein loss during storage and administration.

[0007] 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., mosulatuzumab, gefiruzumab, silvosetumab, or rumotuzumab), a surfactant (e.g., polysorbate 20 (PS20) or poloxamer 188 (P188)), 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 a specific embodiment, the molar ratio of surfactant to therapeutic protein is about 71. In another specific embodiment, the molar ratio of surfactant to therapeutic protein is about 79. In yet another specific embodiment, the molar ratio of surfactant to therapeutic protein is about 48. In other embodiments, the molar ratio of surfactant (e.g., P188) to therapeutic protein 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 therapeutic protein is about 14. In another specific embodiment, the molar ratio of surfactant (e.g., P188) to therapeutic protein is about 11.5.

[0008] In some embodiments, the concentration of the surfactant (e.g., PS20 or P188) is 0.01% to 0.12% weight-by-volume (w / v) (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%, 0.08% to 0.10%, 0.09% to 0.11%, 0.10% 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%, 0.08% to 0.11% (w / v) 0.07%, 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)).

[0009] In some embodiments, the concentration of methionine is 1 mM to 50 mM (e.g., 1 mM to 10 mM, 10 mM to 20 mM, 20 mM to 30 mM, 30 mM to 40 mM, or 40 mM to 50 mM, e.g., about 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 In some embodiments, the concentration of methionine is about 10 mM.

[0010] In some embodiments, the concentration of the buffer 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., about 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).

[0011] In some embodiments, the concentration of the 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., mosulatuzumab, gefizumab, sivusituzumab, or rumatuzumab) 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). In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some embodiments, the concentration of the therapeutic protein is about 1 mg / ml. In some 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.

[0012] 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 concentration of about 1 mg / ml 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., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab) and / or a surfactant (e.g., PS20) in combination with the therapeutic The molar ratio of the 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, for example, 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 specific embodiment, the molar ratio of the surfactant to the therapeutic protein (e.g., mosunitumumab or rumotuzumab) is about 71. In another specific embodiment, the molar ratio of the surfactant to the therapeutic protein (e.g., gefiruzumab) is about 79. In some embodiments, the molar ratio of the surfactant to the therapeutic protein (e.g., rumotuzumab) is about 48.

[0013] In some embodiments, the buffer is histidine, phosphate, succinate, acetate or a combination thereof. For example, in some embodiments, the buffer is histidine, such as histidine acetate or histidine hydrochloride (HCl). The concentration of the buffer (e.g., histidine, such as histidine acetate or histidine HCl, such as L-histidine acetate or L-histidine HCl) can be, for example, 8mM to 12mM, for example, about 8mM, about 9mM, about 10mM, about 11mM or about 12mM. In some embodiments, the concentration of the buffer (e.g., histidine, such as histidine acetate or histidine HCl, such as L-histidine acetate or L-histidine HCl) is about 10mM or 20mM. In a specific embodiment, the buffer is histidine HCl with a concentration of about 20mM. In another specific embodiment, the buffer is histidine acetate with a concentration of about 20mM. In another specific embodiment, the buffer is histidine acetate with a concentration of about 10mM.

[0014] In some embodiments, the pharmaceutical composition further comprises a tonicity agent, such as a sugar, an amino acid, or a salt. In embodiments where the tonicity agent is a sugar, the sugar may be, for example, sucrose, glucose, glycerol, or trehalose. In particular embodiments, the sugar is sucrose. In some embodiments, the concentration of the tonicity agent (e.g., a sugar, such as sucrose) is 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 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 400 mM to 400 mM, 0mM, about 240mM, about 250mM, about 260mM, about 270mM, about 280mM, about 290mM, about 300mM, about 350mM, about 400mM, about 450mM, or about 500mM). In particular embodiments, the concentration of the tonicity agent (eg, a sugar, such as sucrose) is about 240 mM.

[0015] In certain embodiments, the pharmaceutical composition further comprises an antioxidant. In certain embodiments, the antioxidant is N-acetyl-DL-tryptophan. In certain embodiments, the concentration of N-acetyl-DL-tryptophan is 0.1mM to 0.5mM (e.g., about 0.1mM, about 0.15mM, about 0.2mM, about 0.25mM, about 0.3mM, about 0.35mM, about 0.4mM, about 0.45mM or about 0.5mM). In certain embodiments, the concentration of N-acetyl-DL-tryptophan is about 0.3mM.

[0016] In some embodiments, the pharmaceutical composition has a 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, In some embodiments, the pharmaceutical composition has a pH of about 5.1 to 6.1. In a specific embodiment, the pharmaceutical composition has a pH of about 5.8. In another specific embodiment, the pharmaceutical composition has a pH of about 5.5.

[0017] 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 may be a T cell-dependent bispecific antibody (TDB) or a T cell-engaging bispecific antibody (TCB), such as 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) may bind to CD20, FcRH5, or HER2. In some embodiments, the target antigen binding portion is a Fab or a cross-Fab. In some embodiments, the therapeutic protein is mosunetuzumab, gefuzumab, sivostatin, or rumotuzumab.

[0018] In some embodiments, the bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., a T cell-dependent bispecific antibody; e.g., mosulatuzumab) has a methionine at position 257 (as in the EU index) of the Fc region. 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 no greater than about 6% over two weeks at 40° C.

[0019] In another aspect, a pharmaceutical composition includes 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 TDB or TCB; e.g., an anti-CD20 / anti-CD3, anti-FcRH5 / anti-CD3, or anti-HER2 / anti-CD3 TDB or TCB; e.g., mosulatuzumab, gefuzumab, silvosetumab, or rumatuzumab), 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 is at 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., 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, 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 concentration of the surfactant is about 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 concentration of methionine is about 10 mM.

[0020] In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to bispecific antibody (e.g., anti-CD3 bispecific antibody; e.g., T cell-dependent bispecific antibody or T cell-engaging bispecific antibody; e.g., mosulatuzumab, gefizumab, silvosetumab, or rumotuzumab) is 100 or less, e.g., 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 50 or less, 60 or less, 70 or less, 80 or less, 90 or less, 10 ... In some embodiments, the molar ratio of surfactant (e.g., PS20 or P188) to bispecific antibody is 1 to 30, 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, for example, 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 specific embodiment, 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 a certain other specific embodiment, the molar ratio of surfactant to bispecific antibody is about 48. In some embodiments, the surfactant is PS20 and the concentration of PS20 is about 0.06% (w / v).

[0021] In some embodiments, the concentration of the bispecific antibody (e.g., an anti-CD3 bispecific antibody; e.g., TDB or TCB; e.g., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab) is between about 0.1 mg / ml and about 30 mg / ml, e.g., between about 0.1 mg / ml and about 10 mg / ml, between about 0.1 mg / ml and about 5 mg / ml, between about 0.1 mg / ml and about 3 mg / ml, between about 0.1 mg / ml and about 2 mg / ml, between about 0.1 mg / ml and about 1.5 mg / ml, between about 0.3 mg / ml and about 2 mg / ml, between about 0.5 mg / ml and about 2 mg / ml, between about 0.5 mg / ml and about 30 mg / ml. 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, 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, 1 mg / ml to about 30 mg / ml, about 2 mg / ml to about 30 mg / ml, about 3 mg / ml to about 30 mg / ml, about 4 mg / ml to about 30 mg / ml, about 5 mg / ml to about 30 mg / ml, about 6 mg / ml to about 30 mg / ml, about 7 mg / ml to about 30 mg / ml, about 8 mg / ml to about 30 mg / ml, about 9 mg / ml to about 30 mg / 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 1 between about 4 mg / ml and about 30 mg / ml, between about 15 mg / ml and about 30 mg / ml, between about 20 mg / ml and about 30 mg / ml, between about 25 mg / ml and about 30 mg / ml, between about 1 mg / ml and about 20 mg / ml, between about 2 mg / ml and about 20 mg / ml, between about 3 mg / ml and about 20 mg / ml, between about 4 mg / ml and about 20 mg / ml, between about 5 mg / ml and about 20 mg / ml, between about 6 mg / ml and about 20 mg / ml, between about 7 mg / ml and about 20 mg / ml, between about 8 mg / ml and about 20 mg / ml, between about 9 mg / ml and about 20 mg / ml,Between about 10 mg / ml and about 20 mg / ml, between about 11 mg / ml and about 20 mg / ml, between about 12 mg / ml and about 20 mg / ml, between about 13 mg / ml and about 20 mg / ml, between about 14 mg / ml and about 20 mg / ml, between about 15 mg / ml and about 20 mg / ml, between about 1 mg / ml and about 15 mg / ml, between about 2 mg / ml and about 15 mg / ml, between about 3 mg / ml and about 15 mg / ml, about 4 mg / ml Between about 15mg / ml and about 15mg / ml, between about 5mg / ml and about 15mg / ml, between about 6mg / ml and about 15mg / ml, between about 7mg / ml and about 15mg / ml, between about 8mg / ml and about 15mg / ml, between about 9mg / ml and about 15mg / ml, between about 10mg / ml and about 15mg / ml, between about 11mg / ml and about 15mg / ml, between about 12mg / ml and about 15mg / ml, between about 12mg / ml and about 14mg / ml between about 13 mg / ml and about 15 mg / ml, between about 13 mg / ml and about 14 mg / ml, between about 14 mg / ml and about 15 mg / ml, between about 1 mg / ml and about 10 mg / ml, between about 2 mg / ml and about 10 mg / ml, between about 3 mg / ml and about 10 mg / ml, between about 4 mg / ml and about 10 mg / ml, between about 5 mg / ml and about 10 mg / ml, between about 6 mg / ml and about 10 mg / ml, and about 7 mg / ml. In some embodiments, 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.

[0022] 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, for example, 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

[0023] 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.

[0024] In some embodiments, the 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 the 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.

[0025] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising: a 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 as in (a) and a VL domain as in (b). In certain 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. For example, in some embodiments 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, the bispecific antibody is mosunituzumab.

[0026] In some embodiments, the anti-target arm is an anti-CD20 arm comprising a CD20 binding domain, wherein the CD20 binding domain comprises: 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 as in (a) and a VL domain as in (b). In certain 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 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, the bispecific antibody is mosunituzumab.

[0027] In another aspect, the disclosure features 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 concentration of PS20 is 0.01% to 0.12% weight-by-volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-CD20 arm, wherein the anti-CD3 arm comprises a CD3 binding domain 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; 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: NO:14; and the anti-CD20 arm comprises 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.

[0028] 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 as in (a) and a VL domain as in (b). In certain 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. 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 as in (a) and a VL domain as in (b). In certain 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 mosunituzumab.

[0029] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising: a 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 as in (a) and a VL domain as in (b). In certain 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. For example, in some embodiments 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, the bispecific antibody is sivostatin.

[0030] In some embodiments, the anti-target arm is an anti-FcRH5 arm comprising a FcRH5 binding domain, wherein the FcRH5 binding domain comprises: 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 CD79b 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 as in (a) and a VL domain as in (b). In certain embodiments, the VH domain of the CD79b binding domain comprises the amino acid sequence of SEQ ID NO: 63 and the VL domain of the CD79b binding domain comprises the amino acid sequence of SEQ ID NO: 64. For example, in some embodiments wherein the VH domain of the CD79b binding domain comprises the amino acid sequence of SEQ ID NO: 63 and the VL domain of the CD79b binding domain comprises the amino acid sequence of SEQ ID NO: 64, the bispecific antibody is sivostatin.

[0031] In another aspect, the disclosure features 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 concentration of PS20 is 0.01% to 0.12% weight-by-volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-FcRH5 arm, wherein the anti-CD3 arm comprises a CD3 binding domain 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; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: NO:70; and the anti-FcRH5 arm comprises a FcRH5 binding domain, which FcRH5 binding domain comprises: HVR-H1, which comprises the amino acid sequence of SEQ ID NO:57; HVR-H2, which comprises the amino acid sequence of SEQ ID NO:58; HVR-H3, which comprises the amino acid sequence of SEQ ID NO:59; HVR-L1, which comprises the amino acid sequence of SEQ ID NO:60; HVR-L2, which comprises the amino acid sequence of SEQ ID NO:61; and HVR-L3, which comprises the amino acid sequence of SEQ ID NO:62.

[0032] 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 as in (a) and a VL domain as in (b). In certain 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. Additionally or alternatively, in some embodiments, the CD79b 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 as in (a) and a VL domain as in (b). In certain embodiments, the VH domain of the CD79b binding domain comprises the amino acid sequence of SEQ ID NO: 63 and the VL domain of the CD79b 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 sivostatin.

[0033] In some embodiments, the anti-CD3 arm comprises a CD3 binding domain comprising: a 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 as in (a) and a VL domain as in (b). In certain 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. For example, in some embodiments 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, the bispecific antibody is rufazumab.

[0034] In some embodiments, the anti-target arm is an anti-HER2 arm comprising a HER2 binding domain, wherein the HER2 binding domain comprises: 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 as in (a) and a VL domain as in (b). In certain 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. For example, in some embodiments 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, the bispecific antibody is rufazumab.

[0035] In another aspect, the disclosure features 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 concentration of PS20 is 0.01% to 0.12% weight-by-volume (w / v), and the bispecific antibody comprises an anti-CD3 arm and an anti-HER2 arm, wherein the anti-CD3 arm comprises a CD3 binding domain comprising: 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: NO:114; and the anti-HER2 arm comprises a HER2 binding domain, the FcRH5 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.

[0036] 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 as in (a) and a VL domain as in (b). In certain 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. 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 as in (a) and a VL domain as in (b). In certain 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. For example, in some embodiments of the pharmaceutical composition, the bispecific antibody is rumetuzumab.

[0037] In some embodiments, the bispecific antibody comprises a deglycosylation 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.

[0038] In embodiments where the therapeutic protein is an antibody (e.g., a bispecific antibody, e.g., TDB or TCB), the antibody may be an IgG antibody (e.g., an IgG1 antibody or an IgG4 antibody). In particular cases, the antibody is a bispecific antibody and the bispecific antibody 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.

[0039] 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).

[0040] In some embodiments, the 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, the Fab molecule 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 at least one Fab molecule that specifically binds to CD20, the Fab molecule comprising: (a) a heavy chain variable VH domain comprising an amino acid sequence that has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%; e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 43; (b) a light chain variable (VL) domain comprising an amino acid sequence that has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%; e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 44; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the 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.

[0041] In some embodiments, the bispecific antibody comprises at least one Fab molecule that specifically binds to CD3, the Fab molecule comprising 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 at least one Fab molecule that specifically binds to CD3, the Fab molecule comprising: (a) a heavy chain variable VH domain comprising an amino acid sequence that has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%; e.g., 95%, 96%, 97%, 98%, 99% 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 that has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99%; e.g., 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 52; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the 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.

[0042] In another aspect, the disclosure features 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 concentration of PS20 is 0.01% to 0.12% weight-by-volume (w / v), and the bispecific antibody comprises one Fab molecule that specifically binds to CD3 and two Fab molecules that each specifically bind to CD20, wherein the Fab molecule that specifically binds to CD3 comprises a CD3 binding domain comprising: 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: The present invention relates to a Fab comprising an amino acid sequence of SEQ ID NO: 50; 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.

[0043] 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 as in (a) and a VL domain as in (b). In certain embodiments, 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 as in (a) and a VL domain as in (b). In certain embodiments, 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 gefizumab.

[0044] In some embodiments, the bispecific antibody comprises a Fab molecule that specifically binds to CD3, wherein the variable domains of (a) the Fab heavy chain and the light chain are exchanged or (b) the constant domains of the Fab heavy chain and the light chain are exchanged. 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 is replaced by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is replaced by arginine (R) or lysine (K) (according to Kabat numbering), and wherein in the constant domain CH1 of the Fab molecule, the amino acid at position 147 is replaced by glutamic acid (E) (EU numbering), and the amino acid at position 213 is replaced by 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.

[0045] 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 composed of a first and a second subunit that can stably associate; wherein the third Fab molecule according to (c) is the same as the first Fab molecule according to (a); wherein in the constant domain CL of the first Fab molecule according to (a) and the third Fab molecule according to (c), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted by arginine (R) or lysine (R). wherein 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) in the constant domain CH1 of the first Fab molecule according to (a) and the third Fab molecule according to (c); and wherein the first Fab molecule according to (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 according to (b), and the second Fab molecule according to (b) and the third Fab molecule according to (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 according to (d).

[0046] In some embodiments, the bispecific antibody is a humanized antibody. In some embodiments, the bispecific antibody is a chimeric antibody.

[0047] In some embodiments, the bispecific antibody comprises an Fc domain, wherein the Fc domain is an IgG Fc domain. In some embodiments, 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 the 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.

[0048] 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 and CH32 domains each comprise a protrusion or a cavity, and wherein the protrusion or cavity in the CH31 domain can be positioned in the cavity or protrusion of the CH32 domain, respectively. In some embodiments, the CH31 domain and the CH32 domain meet at the interface between the protrusion and the cavity. In some embodiments, the CH21 and CH22 domains each comprise a protrusion or a cavity, and wherein the protrusion or cavity in the CH21 domain can be positioned in the cavity or protrusion of the CH22 domain, respectively. In some embodiments, the CH21 domain and the CH22 domain meet at the interface between the protrusion and the cavity.

[0049] In some embodiments of any of the foregoing embodiments, the CD3 binding domain binds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide. The human CD3 polypeptide or cynomolgus monkey CD3 polypeptide can be, for example, a human CD3 epsilon polypeptide or a cynomolgus monkey CD3 epsilon polypeptide, respectively. Alternatively, the human CD3 polypeptide or cynomolgus monkey CD3 polypeptide can be a human CD3 gamma polypeptide or a cynomolgus monkey CD3 gamma polypeptide, respectively.

[0050] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., a liquid preparation for infusion, a liquid preparation for injection, or a liquid preparation for dilution). In specific embodiments, the pharmaceutical composition is a liquid preparation for dilution. In specific embodiments, the liquid preparation 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 between 20 ml and 40 ml (e.g., between 20 ml and 30 ml, between 30 ml and 40 ml, between 20 ml and 35 ml, between 25 ml and 40 ml, between 25 ml and 35 ml, or between 28 ml and 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 specific 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 between 10 ml and 20 ml (e.g., between 10 ml and 15 ml, between 15 ml and 20 ml, between 13 ml and 20 ml, between 10 ml and 17 ml, between 13 ml and 17 ml, or between 14 ml and 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 specific embodiments, the volume of the liquid formulation for dilution is about 15 ml.

[0051] In another specific embodiment, the liquid formulation for dilution is supplied 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 supplied in a container having a volume of about 2.5 ml. In some embodiments, the volume of the liquid formulation for dilution is between 0.2 ml and 2 ml (e.g., between 0.2 ml and 1.5 ml, between 0.5 ml and 2 ml, between 0.5 ml and 1 ml, or between 0.8 ml and 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 specific embodiments, the volume of the liquid formulation for dilution is about 0.5 ml, about 0.9 ml, or about 1 ml.

[0052] In another specific embodiment, the liquid preparation for dilution is supplied in the container with the volume of about 15ml (for example, about 10ml, about 11ml, about 12ml, about 13ml, about 14ml, about 15ml, about 16ml, about 17ml, about 18ml, about 19ml or about 20ml).In certain embodiments, the liquid preparation for dilution is supplied in the container with the volume of about 10ml.In certain embodiments, the volume for the liquid preparation of dilution is between 4ml to 12ml (for example, between 4ml to 8ml, between 8ml to 12ml, between 4ml to 10ml, between 6ml to 12ml, between 6ml to 10ml or between 7ml to 9ml; For example, about 4ml, about 5ml, about 6ml, about 7ml, about 8ml, about 9ml, about 10ml, about 11ml or about 12ml).In a specific embodiment, the volume for the liquid preparation of dilution is about 8ml.

[0053] In some embodiments, the liquid preparation is used to be diluted with a diluent. In some embodiments, the liquid preparation is used to be diluted with a saline solution. In some embodiments, the liquid preparation is used to be diluted with a normal saline solution. In some embodiments, the normal saline solution comprises sodium chloride (NaCl). In some embodiments, the normal saline solution comprises between 0.1% and 1.5% (e.g., between 0.1% and 1.2%, between 0.3% and 1.5%, between 0.4% and 0.5%, between 0.3% and 1%, between 0.8% and 1%, between 0.85% and 0.95%; for example, 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) of NaCl). In some embodiments, the liquid formulation (eg, comprising an anti-FcRH5 / anti-CD3 bispecific antibody; eg, an anti-FcRH5 / anti-CD3 TDB; eg, silvosumab) is undiluted.

[0054] In some embodiments, the pharmaceutical composition is in a container (e.g., a stainless steel container or a nickel steel alloy container (e.g., ) such as a barrel (e.g., a mini barrel) or a can (e.g., a mini can) or a glass container (e.g., a glass vial or a glass ampoule).

[0055] In some embodiments, the pharmaceutical composition comprises no more than 1,000 particles having a diameter of ≥2 μm per ml (e.g., 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less particles having a diameter of ≥2 μm per ml, such as 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 per ml). In some embodiments, the carrier is water.

[0056] In certain 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 certain 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). In certain embodiments, the pharmaceutical composition is stable through three or more freeze-thaw cycles. In certain embodiments, the pharmaceutical composition is stable at about 25 ° C for about two weeks or more (e.g., about three weeks, about four weeks, about six weeks, about eight weeks, about 10 weeks, about 12 weeks, about 24 weeks, or more). In certain embodiments, the pharmaceutical composition is stable at about 25 ° C for about four weeks or more. In some embodiments, the pharmaceutical composition is stable at -20°C for about 48 months or longer (eg, stable at -20°C for about 48 months, about 60 months, about 72 months, about 84 months, about 96 months, or longer).

[0057] In some embodiments of any aspect and embodiment described above or herein, the pharmaceutical composition has a purity of about 85% or more, e.g., as assessed by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, the purity is about 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, e.g., 85% to 90%, 90% to 95%, or 95% to 100%, e.g., as assessed by SE-HPLC. In specific embodiments, the pharmaceutical composition has a purity of about 90% or more as assessed by SE-HPLC or a purity of about 95% or more as assessed by SE-HPLC. In some embodiments, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC for about 36 months or longer 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 purity as assessed by SE-HPLC for about 36 months or longer at about 5°C, e.g., 85% to 90%, 90% to 95%, or 95% to 100% purity as assessed by SE-HPLC for about 36 months or longer at about 5°C). In certain embodiments, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC at about 5°C for about 42 months or longer (e.g., about 42 months, about 60 months, about 72 months, about 84 months, about 96 months or longer at about 5°C).

[0058] In any of the foregoing aspects and embodiments, the pharmaceutical composition has a purity of about 75% or greater as assessed by 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, 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, about 100% or greater, about 101% or greater, about 102% or greater, about 103% or greater, about 104% or greater, about 105% or greater, about 106% or greater, about 107% or greater, about 108% or greater, about 109% or greater, about 110% or greater, about 111% or greater, about 112% or greater, about 113% or greater, about 114% or greater, about 115% or greater, about 116% or greater, about 117% or greater, about 118% or greater, about 119% or greater, about 120% or greater, about 121% or greater, about 122% or greater, about 123% or greater, In some embodiments, the pharmaceutical composition has a purity of about 80% or more as assessed by non-reducing CE-SDS. For example, in some embodiments, the pharmaceutical composition has a purity of about 85% or more as assessed by non-reducing CE-SDS. In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by non-reducing CE-SDS assay for about 36 months or longer 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 purity, e.g., between 85% and 90%, between 90% and 95%, or between 95% and 100% purity as assessed by non-reducing CE-SDS assay). In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by non-reducing CE-SDS assay for about 42 months or longer 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 purity, e.g., between 85% and 90%, between 90% and 95%, or between 95% and 100% purity as assessed by non-reducing CE-SDS assay).In some embodiments, the non-reducing CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

[0059] In some embodiments, a pharmaceutical composition having any of the above shelf life, purity, or stability characteristics described above is a DS. In other embodiments, a pharmaceutical composition having any of the above shelf life, purity, or stability characteristics described above is a DP. In some embodiments, a pharmaceutical composition having any of the above shelf life or stability characteristics described above is frozen (e.g., stored at a temperature between -80°C and 2°C (e.g., about -40°C or -20°C).

[0060] 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-CD79b / anti-CD3 bispecific antibody; e.g., an anti-CD79b / anti-CD3 TDB; e.g., silvostomumab) 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., a saline solution; e.g., a normal saline solution; e.g., a normal saline solution comprising 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 a saline solution. In some embodiments, the liquid formulation is for dilution with a normal saline solution. In some embodiments, the normal saline solution comprises sodium chloride (NaCl). In some embodiments, the normal saline solution comprises between 0.1% and 1.5% (e.g., between 0.1% and 1.2%, between 0.3% and 1.5%, between 0.4% and 0.5%, between 0.3% and 1%, between 0.8% and 1%, between 0.85% and 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.

[0061] In another aspect, the pharmaceutical composition of any preceding aspect and embodiment is for use as a medicament.

[0062] In another aspect, the pharmaceutical composition of any of the preceding aspects and embodiments is used to treat or delay progression of a cell proliferative disorder in a subject in need thereof (eg, a human subject in need thereof).

[0063] In yet another aspect, the pharmaceutical composition of any preceding aspect and embodiments is used to enhance immune function in a subject suffering from a cell proliferative disorder.

[0064] In certain embodiments, cell proliferative disorders are cancers. In certain embodiments, therapeutic proteins are bispecific antibodies formulated to bind to a CD3 molecule on an immune effector cell and a target molecule on a target cell other than the immune effector cell (e.g., a CD20 molecule on a target cell such as a B cell (e.g., expressed by the target cell). In certain embodiments, bispecific antibodies activate immune effector cells after binding to the CD3 molecule and the target molecule. In certain embodiments, the activated immune effector cells can exert cytotoxic effects and / or apoptotic effects on target cells.

[0065] In some embodiments, for example, wherein the therapeutic protein is a bispecific antibody with 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, 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 of 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 Castleman's disease disease) caused by large B-cell lymphoma, B-cell leukemia, follicular lymphoma (FL), follicular neoplasm in situ, mantle cell lymphoma (MCL), mantle cell tumor in situ, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, variant hairy cell leukemia, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granuloma, plasmablastic lymphoma, and primary effusion lymphoma.In particular 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).

[0066] 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.

[0067] In some embodiments, the therapeutic protein is formulated for administration at a dosage 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., about 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, 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 8 0μg, about 90μg, about 100μg, about 200μg, about 250μg, about 300μg, about 400μg, about 500μg, about 600μg, about 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, about 9mg, about 10mg, about 1 In certain embodiments, the therapeutic protein is a bispecific antibody (e.g., TDB or TCB) formulated for administration to a subject at a dose of about 1 mg to about 60 mg.

[0068] In some embodiments, the pharmaceutical composition (e.g., comprising an anti-CD79b / anti-CD3 bispecific antibody; e.g., an anti-CD79b / anti-CD3 TDB; e.g., silvostomumab) is administered to a subject undiluted (e.g., at a concentration of about 1 mg / ml or about 3 ml / ml). In some embodiments, the pharmaceutical composition is administered to a subject after dilution with a saline solution. In some embodiments, the saline solution is a normal saline solution. In some embodiments, the normal saline solution comprises sodium chloride (NaCl). In some embodiments, the saline solution comprises between 0.1% and 1.5% (e.g., between 0.1% and 1.2%, between 0.3% and 1.5%, between 0.4% and 0.5%, between 0.3% and 1%, between 0.8% and 1%, between 0.85% and 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 specific embodiments, the saline solution comprises 0.45% or 0.9% (w / v) NaCl.

[0069] In some embodiments, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., a TDB or TCB; e.g., mosulatuzumab, gefizumab, sivusituzumab, or rumotuzumab) after dilution with normal saline solution is from 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, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 6 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 particular embodiments, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., a TDB or TCB; e.g., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab) after dilution with normal saline solution 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, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TDB; e.g., mosunituzumab) after dilution with saline solution 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, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TCB; e.g., gefiruzumab) after dilution with saline solution is about 0.1 mg / ml or about 0.6 mg / ml.In certain embodiments, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-FcRH5 / anti-CD3 bispecific antibody; anti-FcRH5 / anti-CD3 TDB; e.g., silvostomumab) after dilution with normal saline solution is about 0.003 mg / ml, 0.03 mg / ml, or 0.3 mg / ml.

[0070] In some embodiments, the subject is co-administered with at least one additional therapeutic agent (e.g., one, two, three, four or more additional therapeutic agents). In some embodiments, the at least one additional therapeutic agent comprises 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); ) 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 human.

[0071] In another aspect, the disclosure features a method of treating or delaying 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.

[0072] In another aspect, the disclosure features a method of enhancing immune function in a subject having a cell proliferative disorder, eg, by administering to the subject an effective amount of the pharmaceutical composition of any of the preceding aspects.

[0073] In certain embodiments, cell proliferative disorders are cancers. In certain embodiments, the therapeutic protein of the pharmaceutical composition administered to a subject is a bispecific antibody formulated to bind to a CD3 molecule on an immune effector cell and a target molecule on a target cell other than the immune effector cell (e.g., a CD20 molecule on a target cell such as a B cell (e.g., expressed by the same)). In certain embodiments, the bispecific antibody activates immune effector cells after binding to the CD3 molecule and the target molecule. In certain embodiments, the activated immune effector cells can exert cytotoxic effects and / or apoptotic effects on target cells.

[0074] In some embodiments, for example, wherein the therapeutic protein is a bispecific antibody with 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, 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 of 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 Castleman's disease disease) caused by large B-cell lymphoma, B-cell leukemia, follicular lymphoma (FL), follicular neoplasm in situ, mantle cell lymphoma (MCL), mantle cell tumor in situ, acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, variant hairy cell leukemia, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granuloma, plasmablastic lymphoma, and primary effusion lymphoma.In particular 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).

[0075] 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.

[0076] In some embodiments, the therapeutic protein (e.g., a bispecific antibody) is administered at a dosage 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, about 70 μg, about 75μg, about 80μg, about 90μg, about 100μg, about 200μg, about 250μg, about 300μg, about 400μg, about 500μg, about 600μg, about 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 8m In certain embodiments, the method comprises administering a therapeutic protein (e.g., a bispecific antibody) to a subject at a dose of about 1 mg to about 60 mg.

[0077] In some embodiments, the subject is co-administered with at least one additional therapeutic agent (e.g., one, two, three, four or more additional therapeutic agents). In some embodiments, the at least one additional therapeutic agent comprises 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 (pembrolizumab), AMG 404, REGN2810 (cemipril 2010); ) and AMP-224 (described in WO2017 / 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 pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, intranasally, intrathecally, or intraventricularly. In some embodiments, the subject is human.

[0078] Unless the context clearly indicates otherwise, the various embodiments can be combined.Unless the context clearly indicates otherwise, the various embodiments can be applied to various aspects of the disclosure.

[0079] Certain embodiments of the present disclosure will become apparent from the following more detailed description of certain preferred embodiments and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 A table summarizing delivery and use considerations for various Phase III drug product (DP) formulations.

[0081] Figure 2Graph showing minimum PS20 concentration (% w / v) as a function of dose and DP protein concentration, as determined by IV bag shake studies in 100 ml PO bags. The x-axis shows DP protein concentration in mg / ml, while the y-axis shows dose in mg.

[0082] Figure 3 Graph showing the effect of various IV bag sizes and amounts of anti-CD20 / anti-CD3 TDB 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 condition on the x-axis (from left to right) represents >5 mg of TDB, the second bar represents 2 mg of TDB, and the third bar represents 1 mg of TDB.

[0083] Figure 4A Graph showing the kinetics of protein oxidation at methionine 257 (Met257) of mosulatuzumab formulated with PS20 at 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) at 40°C.

[0084] Figure 4B Graph showing protein oxidation kinetics at methionine 257 of mosulatuzumab formulated with P188 at 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) at 40°C.

[0085] Figure 4C Graph showing protein oxidation kinetics at methionine 257 of mosulatuzumab formulated with srPS20 at 1 mg / ml (triangles), 10 mg / ml (squares), or 60 mg / ml (diamonds) at 40°C.

[0086] Figure 5 Graph showing the kinetics of protein oxidation at methionine 257 of mosulatuzumab formulated with 30 mM histidine (diamonds) or 10 mM histidine (squares) at 40°C.

[0087] Figure 6 Graph showing the percent oxidation of mosulatuzumab at methionine 257 in various formulations after ambient light exposure up to 300,000 lux-hours (at 5500 lux light intensity). The first bar for each formulation on the x-axis (from left to right) represents time = 0; the second bar represents 24 hours; the third bar represents 54 hours; and the last (fourth) bar represents the dark control.

[0088] Figure 7AGraph showing the kinetics of hydrogen peroxide (H2O2) concentration in various mosulatuzumab compositions stored at 5°C for up to 12 months. The BTCT4465A compositions tested included the composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), the composition + H2O2 (squares), the composition + H2O2 + 2.5 mM methionine (triangles), the composition + 5 mM methionine (dark X-axis), the composition + H2O2 + 5 mM methionine (light X-axis), and the composition + H2O2 + 10 mM methionine (light X-axis). The H2O2 concentration was measured by Red assay measurements.

[0089] Figure 7B Graph showing oxidation kinetics at methionine 257 in various mosulatuzumab compositions stored at 5°C for up to 12 months. Mosulatuzumab compositions tested included composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + H2O2 (squares), composition + H2O2 + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X-axis), composition + H2O2 + 5 mM methionine (light X-axis), and composition + H2O2 + 10 mM methionine (light X-axis). Oxidation was measured by peptide mapping.

[0090] Figure 8A Graph showing the kinetics of hydrogen peroxide (H2O2) concentration in various mosulatuzumab compositions stored at 25°C for up to six months. The mosulatuzumab compositions tested included the composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), the composition + H2O2 (squares), the composition + H2O2 + 2.5 mM methionine (triangles), the composition + 5 mM methionine (dark X-axis), the composition + H2O2 + 5 mM methionine (light X-axis), and the composition + H2O2 + 10 mM methionine (light X-axis). The H2O2 concentration was measured by Red assay measurements.

[0091] Figure 8BGraph showing oxidation kinetics at tryptophan 107 in the CD20 arm of various mosulatuzumab compositions stored at 25°C for up to six months. Mosulatuzumab compositions tested included composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), composition + H2O2 (squares), composition + H2O2 + 2.5 mM methionine (triangles), composition + 5 mM methionine (dark X-axis), composition + H2O2 + 5 mM methionine (light X-axis), and composition + H2O2 + 10 mM methionine (light X-axis). Oxidation was measured by peptide mapping.

[0092] Figure 8C Graph showing oxidation kinetics at methionine 257 for various mosulatuzumab compositions stored at 25°C for up to six months. Mosulatuzumab compositions tested included the composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), the composition + H2O2 (squares), the composition + H2O2 + 2.5 mM methionine (triangles), the composition + 5 mM methionine (dark X-axis), the composition + H2O2 + 5 mM methionine (light X-axis), and the composition + H2O2 + 10 mM methionine (light X-axis). Oxidation was measured by peptide mapping.

[0093] Figure 8D Graph showing the kinetics of high molecular weight species (HMWS) levels as measured by SEC in various mosulatuzumab compositions stored for up to six months at 25° C. Mosulatuzumab compositions tested included the composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), the composition + H2O2 (squares), the composition + H2O2 + 2.5 mM methionine (triangles), the composition + 5 mM methionine (dark X-axis), the composition + H2O2 + 5 mM methionine (light X-axis), and the composition + H2O2 + 10 mM methionine (light X-axis).

[0094] Figure 8EGraph showing the kinetics of low molecular weight species levels measured by mCE-SDS in various mosulatuzumab compositions stored for up to six months at 25° C. Mosulatuzumab compositions tested included the composition alone (1 mg / ml mosulatuzumab, 15 mM histidine acetate, 0.08% (w / v) PS20, 160 mM sucrose, pH 5.8; control, diamonds), the composition + H2O2 (squares), the composition + H2O2 + 2.5 mM methionine (triangles), the composition + 5 mM methionine (dark X-axis), the composition + H2O2 + 5 mM methionine (light X-axis), and the composition + H2O2 + 10 mM methionine (light X-axis).

[0095] Figure 9A Graph showing the change in HMWS levels over time as measured by SE-HPLC in various mosunimotuzumab formulations stored at 40°C and 75% relative humidity (RH) for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0096] Figure 9B Graph showing the change in monomer levels over time as measured by SE-HPLC in various mosunimotuzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0097] Figure 9C Graph showing the change in LMWS levels over time as measured by SE-HPLC in various mosunituzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0098] Figure 10A Graph showing the change in acidic variants as measured by icIEF in various mosunituzumab formulations stored at 40°C and 75% RH over time for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0099] Figure 10B Graph showing the evolution of the main peak over time as measured by icIEF in various mosunimotuzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0100] Figure 10C Graph showing the change in basic variants over time as measured by icIEF in various mosunituzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0101] Figure 11AGraph showing the change in the sum before peak as measured by mCE-SDS over time for various mosunituzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0102] Figure 11B Graph showing the time course of the sum of the main peaks measured by mCE-SDS in various mosunituzumab formulations stored at 40°C and 75% RH for up to one month. Formulations F1 to F5 are characterized in Table 5.

[0103] Figure 12 is a graph showing the Donan effect on the pH value of mosunimotuzumab compositions.

[0104] Figure 13 : Schematic diagram showing the structure of Gefituzumab.

[0105] Figure 14 : Formulation Development GLP Tox and Entry-in-Human Studies. Surfactant content of Formulations F1 to F5, initially and after 6 weeks of storage at 5°C, 25°C or 40°C.

[0106] Figures 15A to 15C : Formulation Development GLP Tox and Entry-in-Human Studies, Size Exclusion Chromatography (SEC) of Formulations F1 to F5, initially and after 6 weeks storage at 5°C, 25°C or 40°C. Figure 15A : Main peak, Figure 15B : high molecular weight (HMW); Figure 15C : Low molecular weight (LMW).

[0107] 16A to 16C : Formulation Development GLP Tox and Entry-in-Human Studies, Ion Exchange Chromatography (IEC) of Formulations F1 to F5, initially and after 6 weeks storage at 5°C, 25°C, or 40°C. Figure 16A : Main peak, Figure 16B :HMW; Figure 16C :LMW

[0108] Figure 17 : Formulation development - 84-week analysis results of Formulation F1. F1 = 5 mg / ml RO7022859 (i.e., Gefituzumab), 20 mM histidine HCl pH 5.5, 240 mM sucrose, 10 mM methionine, 0.05% (w / v) polysorbate 20.

[0109] Figure 18A : Formulation Development GLP Tox and Entry-in-Human Studies, huCD20 binding for Formulations F1 to F5, initially and after 3 and 6 weeks of storage at 5°C, 25°C, or 40°C.

[0110] Figure 18B: Formulation Development GLP Tox and Entry-in-Human Studies, huCD3 binding for Formulations F1 to F5, initially and after 3 and 6 weeks of storage at 5°C, 25°C, or 40°C.

[0111] Figures 19A to 19B : Development studies for Phase III and commercial formulations. After 104 weeks of storage at 5°C, size exclusion (SE)-HPLC% HMWS ( Figure 19A ) and ion exchange (IE)-HPLC% acidic region ( Figure 19B ) as a function of protein concentration.

[0112] FIG. 20A to FIG. 20B : Development studies for Phase III and commercial formulations. After 6 weeks of storage at 40°C, SE-HPLC% HMWS ( Figure 20A ) and IE-HPLC% acidic region ( Figure 20B ) as a function of pH and stabilizer (methionine) addition.

[0113] Figure 21 : Development studies towards Phase III and commercial formulation. Gefituzumab SE-HPLC % HMWS (including visible particle formation) and IE-HPLC % acidic zone as a function of tonicity agent after 26 weeks of storage at 25°C.

[0114] Figure 22 : Development studies towards Phase III and commercial formulation. Gefituzumab SE-HPLC % HMWS (including visible particle formation) and IE-HPLC % acidic zone as a function of surfactant after 7 days of shaking at 25°C.

[0115] Figure 23 : Development studies towards Phase III and commercial formulation. Gefituzumab PS20 content [mg / ml] and visible particle formation as a function of protein concentration, initially and after 104 weeks of storage at 5°C.

[0116] Figure 24 : Long-term stability data: PS20 content of exemplary Gefituzumab DP batches in stability studies (stored at 2°C to 8°C). DETAILED DESCRIPTION

[0117] I. Definition

[0118] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this disclosure belongs. In some cases, for the sake of clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and these definitions included herein should not be construed as indicating a significant difference from the commonly understood meaning in the art compared to the definitions of terms commonly understood in the prior art.

[0119] As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments involving the value or parameter itself.

[0120] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an isolated peptide" refers to one or more isolated peptides.

[0121] Throughout the specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or groups of integers.

[0122] The terms "pharmaceutical composition" and "pharmaceutical formulation" are used interchangeably herein and refer to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0123] The term "therapeutic protein" refers to a protein that can be administered to a subject to elicit a biological response associated with the treatment or improvement of a disease or pathological condition. Therapeutic proteins include biopharmaceuticals 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, e.g., T cell-engaging bispecific antibodies (TCBs), e.g., anti-CD20 / anti-CD3 TCBs, e.g., gefuzumab), immunoconjugates,

[0124] "Pharmaceutically acceptable carrier" or "carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers or vehicles include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0125] The term "shelf life" refers to the length of time a product (e.g., a therapeutic protein (e.g., a bispecific antibody, e.g., a TDB, e.g., an anti-CD20 / anti-CD3 TDB, e.g., mosunetuzumab, e.g., a TCB, e.g., an anti-CD20 / anti-CD3 TCB, e.g., gefiruzumab)) can be stored without becoming unsuitable for use (e.g., use by administration to a subject) or sale. In some embodiments, the shelf life is the length of time during which 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.

[0126] A "stable" pharmaceutical formulation is one in which a protein (e.g., a therapeutic protein) substantially retains its physical stability and / or chemical stability and / or biological activity during storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity during storage (e.g., frozen storage). The shelf life is typically selected based on the expected shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in, for example, Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability within a selected time period can be measured at a selected exposure and / or temperature. Stability can be assessed qualitatively and / or quantitatively in a variety of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); assessing ROS formation (e.g., by using a light stress assay or a 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidizing specific amino acid residues of a protein (e.g., a Met residue of an antibody); assessing charge heterogeneity by using cation exchange chromatography, imaged capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino-terminal or carboxyl-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact polypeptides (e.g., therapeutic proteins); peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or target binding function of a protein (e.g., binding of an antibody to its antigen, such as binding of a TDB to T cells and / or target cells); etc. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and / or Trp oxidation), isomerization (e.g., Asp isomerization), cleavage / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differences in glycosylation, etc.

[0127] A protein (e.g., a therapeutic protein such as a bispecific antibody) in a pharmaceutical formulation "retains its physical stability" if the protein shows no or minimal signs of aggregation, precipitation, fragmentation, and / or denaturation when inspected visually for color and / or clarity, or when measured by UV light scattering or by size exclusion chromatography.

[0128] A protein (e.g., a therapeutic protein, such as a bispecific antibody) in a pharmaceutical formulation "retains its chemical stability" if its chemical stability at a given time is such that the protein (e.g., a therapeutic protein (e.g., a bispecific antibody, such as a TDB or TCB)) is considered 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, such as a TDB or TCB, such as an anti-CD20 / anti-CD3 TDB or TCB). Chemical alterations can involve protein oxidation, which can be assessed using, for example, tryptic peptide mapping, reversed-phase high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC / MS). Other types of chemical alterations include charge changes of a protein (e.g., a therapeutic protein (e.g., a bispecific antibody, such as a TDB or TCB, such as an anti-CD20 / anti-CD3 TDB or TCB), which can be assessed by, for example, ion exchange chromatography or icIEF.

[0129] A protein (e.g., a therapeutic protein (e.g., a bispecific antibody, such as a TDB or TCB)) "retains its biological activity" if its biological activity at a given time is within about 20% (such as within about 10%) of the biological activity exhibited when the pharmaceutical formulation is prepared (within the error of the assay), as determined, for example, in a receptor binding assay.

[0130] As used herein, the "biological activity" of a protein (e.g., a therapeutic protein (e.g., a bispecific antibody, such as a TDB or TCB)) refers to the ability of the protein (e.g., a therapeutic protein (e.g., a bispecific antibody, such as a TDB or TCB)) to bind to its target, for example, the ability of an antibody to bind to its antigen (e.g., the ability of a TDB or TCB to bind to T cells and / or target cells). It may further include a biological response that can be measured in vitro or in vivo. Such activity can be antagonistic or agonistic.

[0131] A protein that is "sensitive to oxidation" (e.g., a therapeutic protein such as a bispecific antibody, e.g., TDB or TCB) is a protein that contains one or more residues that have been found 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.

[0132] The term "percent oxidation" refers to the percentage of a protein (e.g., a therapeutic protein) in a formulation (e.g., a pharmaceutical composition) that is oxidized at a specific amino acid residue (e.g., a Met residue). The percent oxidation can be determined, for example, by mass spectrometry (MS) analysis of one or more tryptic peptides in which one or more specific amino acid residues susceptible to oxidation are present. For example, the percent oxidation can be determined after AAPH stress testing within 9 months, 12 months, 18 months, or two years from the initial production of the protein (e.g., a therapeutic protein) or a pharmaceutical composition thereof.

[0133] As used herein, the term "as assessed by the AAPH stress test" means the percent oxidation at a specific amino acid residue (e.g., a Met residue) as determined by mass spectrometry analysis of tryptic peptides about 24 hours after formulation of a protein (e.g., a therapeutic protein, e.g., 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) at about 40° C. (e.g., in a formulation of about 10 mg / ml 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 percent oxidation.

[0134] As used herein, "buffer" refers to a buffered solution (also referred to herein as "buffer") that resists pH changes through the action of an acid-base conjugate component. In some embodiments, the buffer of the present disclosure has a pH in the range of about 4.5 to about 8. In some embodiments, the buffer 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), for example, about pH 5.5 or 5.8. In a specific embodiment, the pH of the buffer is about 5.5. In another specific embodiment, the pH of the buffer is about 5.8. Exemplary buffers used 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, histidine is histidine acetate or histidine HCl, sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, or a mixture thereof. In a specific embodiment, the buffer is histidine HCl or histidine acetate.

[0135] As used herein, "tonicity agent" refers to an agent that can be added to a liquid (e.g., an aqueous solution) to adjust the tonicity of the liquid. Tonicity refers to the measurement 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 allows 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 (e.g., when administered subcutaneously) by preventing osmotic pressure shock at the application site. Exemplary tonicity agents include carbohydrates (e.g., sucrose, glucose, dextrose, glycerol, glycerol, mannitol, and trehalose), amino acids, and salts (e.g., sodium chloride and potassium chloride).

[0136] As used herein, "surfactant" refers to a surfactant, 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); Sodium lauryl sulfate; sodium lauryl sulfate; sodium octyl glucoside; lauryl, myristyl, linoleyl, or stearyl sulfobetaine; lauryl, myristyl, linoleyl, or stearoyl sarcosine; linoleyl, myristyl, or cetyl betaine; lauramidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmitoylpropyl, or isostearamidopropyl betaine (e.g., lauroyl propyl); myristamidopropyl, palmitoylpropyl, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl-taurate; and MONAQUAT TM series (Mona Industries, Inc., Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Block copolymers, e.g. F-68); etc. In one embodiment, the surfactant herein is polysorbate 20 (PS20). In another embodiment, the surfactant herein is poloxamer 188 (P188).

[0137] "Preservatives" are compounds that can be optionally included in a formulation to substantially reduce bacterial action therein, thereby promoting, for example, the production of multi-purpose formulations. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethylammonium 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, butanol, and benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and meta-cresol. In one embodiment, the preservative herein is benzyl alcohol. In some embodiments, the formulation does not include a preservative.

[0138] The "surfactant to therapeutic protein molar ratio" (surfactant:protein) is the ratio of surfactant to therapeutic protein, where each component is expressed in molar concentrations (also called molar concentrations). This ratio is described by equation (1):

[0139]

[0140] As used herein, "drug substance" or "DS" refers to a pharmaceutical composition that is formulated for storage (e.g., frozen storage) prior to administration to a subject. The DS may have a concentration of therapeutic protein that is greater than the concentration of the therapeutic protein to be administered to a subject. Thus, in some cases, the DS is diluted prior to administration to a subject.

[0141] As used herein, "drug product" or "DP" refers to a pharmaceutical composition in its final configuration so that it is ready for administration to a subject (e.g., in a final vial configuration). The concentration of the therapeutic protein in the DP may be the concentration to be administered to a subject. Alternatively, if the DP is administered with a diluent, or if it is intended to be administered in combination with other therapeutic agents, the concentration of the DP may be higher than the concentration to be administered to a subject.

[0142] Throughout this application, unless otherwise indicated, the techniques used can be found in 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 Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0143] Appropriately, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters, unless otherwise indicated. Therefore, before describing the methods and uses of the present invention, it should be understood that the present disclosure is not limited to the specific methods, protocols, cell lines, animal species or genera, constructs, and reagents described, which can, of course, vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0144] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly connected by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain with two or more amino acids, rather than the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to chains with two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of any of these terms, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modification products of the polypeptide, which include but are not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protection / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, and is not necessarily translated from a specified nucleic acid sequence. It can be generated in any way, including by chemical synthesis. The size of the polypeptides of the present disclosure can be 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, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded; and polypeptides that do not have a defined three-dimensional structure, but can adopt a large number of different conformations, are referred to as unfolded.

[0145] An "isolated" polypeptide or variant or derivative thereof is intended to mean a polypeptide that is not in its native environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells are considered isolated for the purposes of the present invention, as are natural or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0146] The term "antibody" herein is used in the broadest sense and encompasses various 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 intended antigen-binding activity (e.g., antigen-binding fragments of an antibody).

[0147] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments thereof.

[0148] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody and bind to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen fragments.

[0149] "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., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), an antibody fragment or a portion thereof (e.g., a Fab fragment, Fab'2, scFv antibody, SMIP, domain antibody, diabody, miniantibody, scFv-Fc, affibody, nanobody, and VH domain and / or VL domain of an antibody), a receptor, a ligand, an aptamer, and other molecules with an identified binding partner.

[0150] As used herein, the term "antigen binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding portion is capable of directing the entity to which it is attached (e.g., a cytokine or a second antigen binding portion) to a target site, for example, to a specific type of tumor cell or tumor stroma with an antigenic determinant. The antigen binding portion includes antibodies and fragments thereof as further defined herein. Preferred antigen binding portions include the antigen binding domains of antibodies, which include antibody heavy chain variable regions and antibody light chain variable regions. In certain embodiments, the antigen binding portion may include antibody constant regions as further defined herein and as known in the art. Available heavy chain constant regions include any one of the following five isotypes: α, δ, ε, γ or μ. Available light chain constant regions include any one of the following two isotypes: κ and λ.

[0151] "Cross" Fab molecules (also referred to as "Crossfab") refer to Fab molecules in which the variable domains or constant domains of the Fab heavy chain and Fab light chain are exchanged (i.e., replaced with each other), i.e., the crossover Fab molecules comprise a peptide chain (VL-CH1, N-terminal to C-terminal direction) consisting of a light chain variable domain VL and a heavy chain constant domain 1CH1, and a peptide chain (VH-CL, N-terminal to C-terminal direction) consisting of a heavy chain variable domain VH and a light chain constant domain CL. For clarity, in the crossover Fab molecules in which the variable domains of the Fab light chain and the variable domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in the crossover Fab molecules in which the constant domains of the Fab light chain and the constant domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0152] In contrast, by a "conventional" Fab molecule is meant a Fab molecule in its natural form, i.e., comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH1, in N-terminal to C-terminal direction) and a light chain consisting of a light chain variable domain and a constant domain (VL-CL, in N-terminal to C-terminal direction).

[0153] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0154] (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));

[0155] (b) CDRs occurring 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);

[0156] (c) antigenic 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

[0157] (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).

[0158] 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.

[0159] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using the VH or VL domains from antibodies that bind to the antigen, respectively, to screen for libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0160] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the 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) in the Fc region may be present or absent. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described above by Kabat et al.

[0161] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0162] "Percent amino acid sequence identity (%)" or "percent sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of sequence identity. Alignment for determination of percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity%. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the US Copyright Office, Washington DC, 20559, along with user documentation, where it is registered with 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 the source code. The ALIGN-2 program should be compiled into Operating system (including digital All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0163] When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can alternatively be expressed as the % amino acid sequence identity that a given amino acid sequence A has or contains with a given amino acid sequence B) is calculated as follows:

[0164] Multiply 100 by the fraction X / Y

[0165] 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 where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0166] 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.

[0167] A "human consensus framework" is a framework that represents the amino acid residues that are most commonly found in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the subset of sequences is a subset as described by Kabat et al., supra. In one embodiment, for VL, the subset is subgroup kappa I as described by Kabat et al., supra. In one embodiment, for VH, the subset is subgroup III as described by Kabat et al., supra.

[0168] "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 will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody optionally may 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.

[0169] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure, or has heavy chains that contain an Fc region as defined herein.

[0170] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or the amino acid sequence of an antibody derived from a non-human source using a full repertoire of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced 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). Methods for preparing human monoclonal antibodies can also be 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 to an antigen a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, e.g., immunizing a xenogeneic mouse (see, e.g., for XENOMOUSE). TM (See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) for human antibodies generated by human B cell hybridoma technology.)

[0171] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (for example, containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are generally present in small amounts), the individual antibodies comprising the population have identity and / or bind identical epitopes. In contrast to polyclonal antibody preparations typically comprising different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody production by any ad hoc method. For example, the monoclonal antibody to be used according to the present disclosure can be prepared 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 human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0172] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical compositions.

[0173] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 146,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ).

[0174] As used herein, the term "half antibody" refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain.

[0175] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0176] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as 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.

[0177] As used herein, the term "cluster of differentiation 3" or "CD3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys (cynomolgus)) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, 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 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length; human CD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length, cynomolgus monkey CD3ε protein (NCBI RefSeq No. NP_001270544.1), which is 198 amino acids in length; and cynomolgus monkey CD3γ protein (NCBI RefSeq No. NP_001270839.1), which is 181 amino acids in length.

[0178] Unless otherwise indicated, the term "cluster of differentiation 20" or "CD20" as used herein 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 includes "full-length," unprocessed CD20, as well as any form of CD20 produced by processing in cells. The term also encompasses naturally occurring variants of CD20, including, for example, splice variants or allelic variants. CD20 includes, for example, human CD20 protein (see, e.g., NCBI RefSeq Nos. NP_068769.2 and NP_690605.1), which is 297 amino acids in length and can be produced, for example, from variant mRNA transcripts lacking a portion of the 5'UTR (see, e.g., NCBI RefSeq No. NM_021950.3) or longer variant mRNA transcripts (see, e.g., NCBI RefSeq No. NM_152866.2).

[0179] As used herein, the term "FcRH5" or "fragment crystallizable receptor-like 5" refers to any native FcRH5 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes "full-length," unprocessed FcRH5, as well as any form of FcRH5 resulting from processing in cells, unless otherwise indicated. The 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-ProtID: Q96RD9.3), which is 977 amino acids in length.

[0180] Unless otherwise indicated, the term "HER2" as used herein refers to any natural HER2 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed HER2, as well as any form of HER2 produced by processing in cells. The term also encompasses naturally occurring variants of HER2, including, for example, splice variants or allelic variants. HER2 includes, for example, human HER2 protein (see, for example, NCBI RefSeq numbering NP_001276865), which is 1240 amino acids in length. Domain IV of HER2 is an extracellular protein region located closest to the cell membrane. Domain IV has the amino acid sequence of SEQ ID NO:17.

[0181] The terms "anti-CD20 / anti-CD3 antibody," "anti-CD20 / anti-CD3 antibody," "anti-CD20 / anti-CD3 TDB," and "anti-CD20 / anti-CD3 TCB," or variations thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that binds to CD20 and CD3 with sufficient affinity to render the antibody useful as a diagnostic and / or therapeutic agent targeting CD20 and / or CD3. In one embodiment, the extent of binding of the anti-CD20 / anti-CD3 antibody to unrelated, non-CD3 proteins and / or non-CD20 proteins is less than about 10% of the extent of binding of the antibody to CD3 and / or CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody binds to each of CD20 and / or CD3 with 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 smaller, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (KD ). In certain embodiments, the anti-CD20 / anti-CD3 antibody binds to a CD3 epitope conserved between CD3 from different species and / or a CD20 epitope conserved between CD20 from different species. The anti-CD20 / anti-CD3 bispecific antibodies that can be used in the methods of the present disclosure include any anti-CD20 / anti-CD3 bispecific antibodies described in PCT Publication No. WO 2015 / 095392, the entire text of which is incorporated herein by reference in its entirety. In some cases, the anti-CD20 / anti-CD3 bispecific antibody is an anti-CD20 / anti-CD3 T cell-dependent bispecific antibody (TDB). In specific embodiments, the anti-CD20 / anti-CD3 bispecific antibody is mosunetuzumab (also known as BTCT4465A or RG 7828), as defined in International Nonproprietary Name (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., 2+1 TCB) described in U.S. Patent No. 9,914,776, which is incorporated herein by reference in its entirety. In a specific embodiment, the anti-CD20 / anti-CD3 bispecific antibody is Gefituzumab (also known as RO 7082859 or RG6026), as defined in International Nonproprietary Name (INN) List 121 (WHO Drug Information, Recommended INN: List 83, 2020, Vol. 34, No. 1, p. 39), and CAS Registry Number 2229047-91-8.

[0182] The terms "anti-CD79b / anti-CD3 antibody," "anti-CD79b / anti-CD3 bispecific antibody," "anti-CD79b / anti-CD3 TDB," or variations thereof refer to a multispecific antibody (e.g., a bispecific antibody) that binds to CD79b and CD3 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting CD79b and / or CD3. In one embodiment, the extent of binding of the anti-CD79b / anti-CD3 bispecific antibody to unrelated, non-CD3 proteins and / or non-CD79b proteins is less than about 10% of the extent of binding of the antibody to CD3 and / or CD79b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-CD79b / anti-CD3 bispecific antibody binds to each of CD79b and / or CD3 with 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 smaller, for example, 10 -8 M to 10-13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K D In certain embodiments, the anti-CD79b / anti-CD3 antibody binds to a CD3 epitope that is conserved between CD3s from different species and / or a CD79b / anti-CD3 epitope that is conserved between CD79b / anti-CD3s from different species. Anti-CD79b / anti-CD3 bispecific antibodies useful in the methods of the present disclosure include any of the anti-CD79b / anti-CD3 bispecific antibodies described in PCT Publication Nos. WO 2016 / 205520, WO 2015 / 095392, and WO 2014 / 210064, and U.S. Pat. No. 10,323,094, which are incorporated herein by reference in their entireties. In some cases, the anti-CD79b / anti-CD3 bispecific antibody is an anti-CD79b / anti-CD3 T cell-dependent bispecific antibody. In a specific embodiment, the anti-FcRH5 antibody is sivosetumab (also known as BFCR4350A or RO7187797), which is described in WHO Drug Information (International Nonproprietary Name), Recommended INN: List 84, Volume 34, Issue 3, 2020 (see page 701), and has a CAS Registry Number of 2249888-53-5. Sivosetumab is an Fc-engineered, humanized, full-length, non-glycosylated IgG1κ T cell-dependent bispecific antibody (TDB) that binds to FcRH5 and CD3.

[0183] The terms "anti-HER2 / anti-CD3 antibody," "anti-HER2 / anti-CD3 bispecific antibody," "anti-HER2 / anti-CD3 TDB," or variations thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that binds to HER2 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting HER2 and / or CD3. In one embodiment, the extent of binding of the anti-HER2 / anti-CD3 bispecific antibody to unrelated, non-CD3 proteins and / or non-HER2 proteins is less than about 10% of the extent of binding of the antibody to CD3 and / or HER2, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the anti-HER2 / anti-CD3 bispecific antibody binds to each of HER2 and / or CD3 with 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 smaller, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K D). In certain embodiments, the anti-HER2 / anti-CD3 antibody binds to a CD3 epitope conserved between CD3 from different species and / or a HER2 epitope conserved between HER2 from different species. The anti-HER2 / anti-CD3 bispecific antibodies that can be used in the methods of the present disclosure include any anti-HER2 / anti-CD3 bispecific antibodies described in PCT Publication No. WO 2015 / 095392, which is incorporated herein by reference in its entirety. In some cases, 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 rumoxetine, as defined in International Nonproprietary Name (INN) List 124 (WHO Drug Information, Vol. 34, No. 4, 2020, p. 1031), and CAS Registry Number is 2361325-98-4.

[0184] Unless otherwise indicated, the term "cluster of differentiation 79b" or "CD79b" as used herein refers to any natural CD79b from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed CD79b, as well as any form of CD79b produced by processing in cells. The term also encompasses naturally occurring variants of CD79b, including, for example, splice variants or allelic variants. CD79b includes, for example, human CD79b protein (NCBI RefSeq No. NP_000617), which is 229 amino acids in length.

[0185] 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 can be used as a diagnostic and / or therapeutic agent targeting CD79b. In one embodiment, the extent of binding of the anti-CD79b antibody to unrelated, non-CD79b proteins is less than about 10% of the extent of binding of the antibody to CD79b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD79b 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 lower, such as 10 -8 M to 10 -13 M, for example 10 -9 to 10 -13 The dissociation constant (K DIn certain embodiments, the anti-CD79b antibody binds to an epitope of CD79b that is conserved among CD79bs from different species.

[0186] As used herein, the term "vepotuzumab" refers to an anti-CD79b antibody drug conjugate with IUPHAR / BPS number 8404, KEGG number D10761, or CAS registration number 1313206-42-6. Vepotuzumab-piiq may also be referred to interchangeably as "polatuzumab vedotinpiiq," "huMA79bv28-MC-vc-PAB-MMAE," "DCDS4501A," or "RG7596."

[0187] In some embodiments, an antibody drug conjugate comprising the formula:

[0188]

[0189] wherein Ab is vepotuzumab, and wherein p is between 1 and 8. In some embodiments, the anti-CD79b antibody drug conjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the antibody drug conjugate is vepotuzumab-piiq (CAS Reg. No. 1313206-42-6).

[0190] In some embodiments, the antibody drug conjugate is vepotuzumab-piiq, 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, vepotuzumab-piiq has the following structure: immunoglobulin G1-κ auristatin E conjugate, anti-[homo sapiens CD79B (immunoglobulin-associated CD79β)], ​​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 IGHG1*03 (CH1 R120>K(214)(121-218), hinge (219-233), CH2(234-343), CH3(344-448), CHS(449-450))(121-450)], (220-218')-disulfide bond (if unconjugated), with kappa light chain (1'-218') [humanized V-κ (Homo sapiens IGKV1-39*01(80.00%)-IGKJ1* 01)[11.3.9](1'-112')-Homo sapiens IGKC*01(113'-218')]; dimer (226-226":229-229")-disulfide bond; conjugated to monomethyl auristatin E (MMAE) with an average of 3 and 4 cysteinyl groups via a cleavable maleimidocaproyl-pentanoyl-citrullinyl-p-aminobenzylcarbamate (mc-val-cit-PABC) linker.

[0191] A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0192] As used herein, "administering" means a method of administering a dose of a therapeutic protein (e.g., TDB or TCB) or composition (e.g., a pharmaceutical composition, such as a pharmaceutical composition comprising a therapeutic protein (e.g., TDB or TCB) of the present disclosure) to a subject. For example, the pharmaceutical composition used in the methods described herein can be administered, for example, intravenously, subcutaneously, intracutaneously, intramuscularly, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intracapsularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly soaking target cells, by catheter, by lavage, in the form of an emulsion, or in the form of a lipid composition. The method of administration can vary depending on a variety of factors (e.g., the pharmaceutical composition being administered and the severity of the condition, disease, or disorder to be treated).

[0193] As used herein, "a week" is 7 days ± 2 days.

[0194] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the pharmaceutical compositions of the present disclosure are used to delay the development of the disease or slow the progression of the disease.

[0195] 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 comprises 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 such enhancement are known to those of ordinary skill in the art.

[0196] As used herein, "enhancing T cell function" means inducing, causing or stimulating T cells to have a sustained or amplified biological function, or restoring or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include: relative to the level before intervention, increased secretion of gamma interferon from CD8+ T cells, increased proliferation, increased antigen reactivity (e.g., virus, pathogen or tumor clearance). In some embodiments, the enhanced level is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%. The way to measure this enhancement is known to those of ordinary skill in the art.

[0197] As used herein, "delaying progression" of a condition or disease means slowing, hindering, slowing, postponing, stabilizing, and / or postponing the development of a disease or condition (e.g., a cell proliferative disorder, e.g., cancer). Such a delay can be of varying lengths of time, depending on the medical history and / or the individual being treated. It will be apparent to one skilled in the art that a substantial or significant delay can actually encompass prevention, as the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, can be delayed.

[0198] "Reduce" or "inhibit" means the ability to cause an overall decrease, for example, an overall decrease of 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more. In certain embodiments, reduction or inhibition may involve antibody utility functions mediated by the Fc region of the antibody, such utility functions specifically including complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP).

[0199] A "disorder" is any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder.

[0200] The terms "cell proliferative disorder" and "proliferative disorder" refer to conditions 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.

[0201] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric / stomach cancer (including gastrointestinal cancer and gastrointestinal stromal tumor cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic cancer, The present invention relates to carcinoma, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with macular degeneration, edema (such as that associated with brain tumors), Meig's syndrome, brain cancer, and head and neck cancer, and related metastases. In certain embodiments, cancers suitable for 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, 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 cancer, gastric cancer, colorectal cancer (CRC) and hepatocellular carcinoma. Again, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, gastric cancer, glioblastoma and breast cancer, including metastatic forms of those cancers.In other embodiments, the cancer is selected from the group of mature B-cell cancers excluding Hodgkin's lymphoma but including 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 macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BURKISH'S LYMPHOMATOLYSIS LASIK), and leukemia. lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, unclassifiable splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, variant hairy cell leukemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T cell / histiocyte-rich large B cell lymphoma, primary CNS DLBCL, primary cutaneous DLBCL, leg type, EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granuloma, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma: 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 classic Hodgkin lymphoma.

[0202] The term "HER2-positive" cancer includes cancer cells whose HER2 levels are higher than normal levels. Examples of HER2-positive cancers include HER2-positive breast cancer and HER2-positive gastric cancer. Optionally, HER2-positive cancer has an immunohistochemistry (IHC) score of 2+ or 3+ and / or an in situ hybridization (ISH) amplification ratio of ≥2.0. In some cases, HER2-positive cancer is a HER2-positive solid tumor. Additionally or alternatively, HER2-positive cancer can be a locally advanced or metastatic HER2-positive cancer. In some cases, HER2-positive cancer is HER2-positive breast cancer or HER2-positive gastric cancer. In certain embodiments, 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 cancer), 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.

[0203] The term "FcRH5-positive cells" refers to cancers comprising cells that express FcRH5 on their surface. For the purpose 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, for example, using antibodies to FcRH5 in 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 myeloid leukemia (CML), FcRH5-positive chronic myelomonocytic leukemia, FcRH5-positive myelomonocytic leukemia, FcRH5-positive myelomonocytic leukemia, FcRH5-positive myeloid leukemia (CML), FcRH5-positive myelomonocytic leukemia, FcRH5-positive myeloid leukemia (CML), FcRH5-positive myelomonocytic leukemia (CML), FcRH5-positive myelomonocytic leukemia (CML), FcRH5-positive myelomonocytic leukemia (CML), FcRH5-positive myelomonocytic leukemia (CML), FcRH5-positive myeloid ... RH5-positive acute promyelocytic leukemia (APL), FcRH5-positive myeloproliferative disorders, FcRH5-positive platelet-derived 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 mast cell disease, FcRH5-positive mast cell leukemia, FcRH5-positive mast cell sarcoma, FcRH5-positive myeloid sarcoma, FcRH5-positive lymphocytic leukemia, and FcRH5-positive undifferentiated leukemia.

[0204] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. As referred to herein, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive.

[0205] As used herein, the term "tumor antigen" is understood to mean those antigens that are presented on tumor cells. These antigens can be presented on the cell surface using an extracellular portion, which is typically combined with the transmembrane and cytoplasmic portions of the molecule. These antigens can sometimes only be presented by tumor cells, but not by normal cells. Tumor antigens may be expressed only on tumor cells, or may represent tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. More common are tumor antigens presented by both tumor cells and normal cells, and they are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be available for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue.

[0206] "Effect functions" refer to those biological activities attributed to the Fc region of an antibody, which vary with 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0207] As used herein, the term "effector cell" refers to a population of lymphocytes that display effector moiety receptors (e.g., cytokine receptors) and / or Fc receptors on their surfaces, through which they bind to effector moieties (e.g., cytokines) and / or the Fc region of antibodies and contribute to the destruction of target cells, such as tumor cells. Effector cells can, for example, mediate cytotoxic or phagocytic responses. Effector cells include, but are not limited to, effector T cells, such as CD8 T cells. + Cytotoxic T cells, CD4 + Helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0208] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of polypeptides comprising the Fc domain subunits with the same polypeptide to form homodimers. As used herein, modifications that promote association particularly include separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications are complementary to each other so as to promote the association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both of the Fc domain subunits so as to favor their association sterically or electrostatically, respectively. Thus, (heterologous) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding moieties) fused to each subunit are inconsistent. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, the association-promoting modifications comprise individual amino acid mutations, particularly amino acid substitutions, of each of the two subunits of the Fc domain.

[0209] "Activating Fc receptors" are Fc receptors that, upon engagement by the Fc region of an antibody, trigger signaling events that stimulate cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0210] As used herein, the terms "engineering," "engineered," and "engineered" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods. "Engineering" (particularly with the prefix "glycosyl") 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 a cell. In addition, glycosylation engineering includes the effects of mutations and the cellular environment on glycosylation. In one embodiment, glycosylation engineering is a change in glycosyltransferase activity. In a specific embodiment, 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 that have been manipulated to express increased 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 that have been manipulated to express increased 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 that have been manipulated to express reduced levels of α(1,6) glycofucosyltransferase).

[0211] 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 primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny with the same function or biological activity as screened or selected in the original transformed cells are included herein. A host cell is any type of cell system that can be used to produce a protein for use in the present disclosure. In one embodiment, the host cell is engineered to allow the production of antibodies with modified oligosaccharides. In certain embodiments, the host cell has been manipulated to express increased levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cell has been further manipulated to express increased levels of one or more polypeptides having α-mannosidase II (ManII) activity. Host cells include cultured cells, for example, mammalian cultured cells, such as CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name a few, but also include transgenic animals, transgenic plants or cells contained in cultured plant or animal tissues.

[0212] As used herein, the term "polypeptide having GnTIII activity" refers to a β-linked mannoside capable of catalyzing the addition of N-acetylglucosamine (GlcNAc) residues in a β-1,4 linkage to the trimannosyl core of an N-linked oligosaccharide. The term includes fusion polypeptides that exhibit enzymatic activity (with or without dose dependence, as measured in a specific biological assay) similar to, but not necessarily identical to, the activity of β(1,4)-N-acetylglucosaminyltransferase III (also known as β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (EC 2.4.1.144), according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)). Where dose dependence does exist, it need not be identical to that of GnTIII, but rather substantially similar to the dose dependence of a given activity compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or no more than about 25-fold less, and preferably no more than about ten-fold less, and most preferably no more than about three-fold less activity relative to 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 of generating such fusion polypeptides and using them to produce antibodies with increased effector function are disclosed in WO 2004 / 065540, US Provisional Patent Application No. 60 / 495,142, and US Patent Application Publication No. 2004 / 0241817, the contents of which are expressly incorporated herein by reference in their entireties.

[0213] 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 its location in the Golgi complex. Typically, the localization domain comprises the amino terminal "tail" of the enzyme.

[0214] As used herein, the term "polypeptide having ManII activity" refers to a polypeptide that is capable of catalyzing the hydrolysis of terminal 1,3 and 1,6 linked α-D mannose residues in branched GlcNAcMan5GlcNAc2 mannose intermediates of N-linked oligosaccharides. The term includes polypeptides that exhibit enzymatic activity similar to, but not necessarily identical to, the activity of Golgi α-mannosidase II (also known as mannosyl oligosaccharide 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)).

[0215] An "effective amount" of a pharmaceutical composition (e.g., a pharmaceutical composition comprising a therapeutic protein) is the minimum amount required to show at least the expected therapeutic or preventive result, such as a measurable improvement in a particular disorder (e.g., a cell proliferative disorder, such as cancer). The effective amount herein can vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit the expected response in the individual. An effective amount is also an amount in which the beneficial effects of the treatment outweigh any toxic or deleterious effects of the treatment. For prophylactic uses, beneficial or expected results include results such as reducing the risk of disease, lessening severity, or delaying the onset of disease (e.g., delaying the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease). For therapeutic uses, beneficial or expected results include clinical results, such as reducing one or more symptoms caused by the disease, improving the quality of life of the patient, reducing the dose of other drugs required to treat the disease, enhancing the effects of other drugs (such as by targeting, slowing disease progression, and / or prolonging survival). In the case of cancer or tumors, an effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down or expect to stop to some extent) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down and expect to stop to some extent) tumor metastasis; inhibit the growth of the tumor to some extent; and / or alleviate to some extent one or more symptoms associated with the disease. An effective amount can be administered once or multiple times. For the purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly prevent or treat. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and an effective amount of a single agent may be considered if the desired result can be obtained or achieved in combination with one or more other agents.

[0216] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0217] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners to eliminate T cell dysfunction caused by signal transduction on the PD-1 signaling axis, resulting in the restoration or enhancement of 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.

[0218] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the signal transduction generated by the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the 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 and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with the signal transduction generated by the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist can reduce the negative co-stimulatory signal mediated by or through the signal transduction of PD-1 mediated by cell surface proteins expressed on T lymphocytes, thereby making the dysfunctional T cells less dysfunctional (e.g., improving the response of effectors 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). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab) described herein. In another specific aspect, the PD-1 binding antagonist is AMG 404 described herein. In another specific aspect, the PD-1 binding antagonist is REGN2810 (cemipril 2810) described herein. In another specific aspect, the PD-1 binding antagonist is AMP-224 as described herein and in PCT Publication No. WO 2017 / 058780.

[0219] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the signaling generated by the interaction of PD-L1 with one or more of its binding partners (such as 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 partner. In a specific aspect, a PD-L1 binding antagonist inhibits 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, eliminate or interfere with the signaling generated by the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, a PD-L1 binding antagonist can reduce the negative co-stimulatory signal mediated by or through the signaling of PD-L1 mediated by cell surface proteins expressed on T lymphocytes, thereby making dysfunctional T cells less dysfunctional (e.g., improving the response of effectors 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) described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 (BMS-936559) described herein and in PCT Publication No. WO 2016 / 201425. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736 (durvalumab) described herein.

[0220] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the signaling generated by the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, eliminate or interfere with the signaling generated by the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one embodiment, a PD-L2 binding antagonist can reduce the negative co-stimulatory signal mediated by or through the signaling of PD-L2 mediated by cell surface proteins expressed on T lymphocytes, thereby making dysfunctional T cells less dysfunctional (e.g., improving the response of effectors to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0221] II. Pharmaceutical Compositions

[0222] The present disclosure provides pharmaceutical compositions comprising therapeutic proteins such as 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., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab), and uses thereof, e.g., for treating cell proliferative disorders (e.g., cancers, such as 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., mosulatuzumab, gefuzumab, silvosetumab, or rumatuzumab).

[0223] Any therapeutic protein described herein (e.g., an anti-CD3 bispecific antibody; e.g., TDB or TCB; e.g., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab) can be used in the pharmaceutical composition, but it will be understood that other therapeutic proteins can also be used.

[0224] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutic protein, a surfactant (eg, polysorbate 20 (PS20)), a stabilizer (eg, 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 a specific embodiment, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., mosunituzumab or rumotuzumab) is about 71. In another specific embodiment, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., gefizumab) is about 79. In yet another specific embodiment, the molar ratio of surfactant (e.g., PS20) to therapeutic protein (e.g., silvostomumab) is about 48. In other embodiments, the molar ratio of surfactant (e.g., P188) to therapeutic protein is 5 to 50, 5 to 25, 10 to 15, or 15 to 20, for example, 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 (e.g., P188) to therapeutic protein is about 11.5.

[0225] The pharmaceutical composition can have any suitable concentration of the 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., mosulatuzumab, gefiruzumab, sivusituzumab, or rumatuzumab). For example, in any of the foregoing pharmaceutical compositions, the concentration of the therapeutic protein can be from 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 , 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.

[0226] 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 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, 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 / 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 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 anti-CD20 / anti-CD3 TDB is about 1 mg / ml. In a specific embodiment, the concentration of anti-CD20 / anti-CD3 TDB is about 2 mg / ml.

[0227] In some embodiments, the therapeutic protein is an anti-HER2 / anti-CD3 TDB and the concentration of the 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. In some embodiments, the concentration of the 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 the anti-HER2 / anti-CD3 TDB is about 0.5 mg / ml to about 1.5 mg / ml. In specific embodiments, the concentration of the anti-HER2 / anti-CD3 TDB is about 1 mg / ml. In another specific embodiment, the concentration of the anti-HER2 / anti-CD3 TDB is about 2 mg / ml.

[0228] In some embodiments, the therapeutic protein is an anti-CD79b / anti-CD3 TDB and the concentration of the anti-CD79b / 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 the anti-CD79b / cdcRHS / 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 the anti-CD79b / cdcRHS / anti-CD3 TDB is about 0.5 mg / ml to about 3 mg / ml. In certain embodiments, the concentration of the anti-CD79b / cdcRHS / anti-CD3 TDB is about 1 mg / ml. In certain embodiments, the concentration of the anti-FcRH5 / anti-CD3 TDB is about 1.5 mg / ml. In certain embodiments, the concentration of the anti-FcRH5 / anti-CD3 TDB is about 3 mg / ml.

[0229] In some embodiments, the therapeutic protein is an 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 the 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 the anti-CD20 / anti-CD3 TCB is about 0.5 mg / ml to about 1.5 mg / ml. In certain embodiments, the concentration of the anti-CD20 / anti-CD3 TCB is about 1 mg / ml.

[0230] The disclosed pharmaceutical compositions include a surfactant. Any suitable surfactant can be used. In some embodiments, the surfactant is a nonionic surfactant (e.g., a polysorbate (polyoxyethylene (n) sorbitan monolaurate), a poloxamer, a polyoxyethylene alkyl ether, an 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®). For example, Super Refined TM PS20 (PS20 that has been subjected to a proprietary flash chromatography process to obtain higher purity and is available from Avantor Performance Materials, LLC (Center Valley, PA, US)) or polysorbate 80 (PS80; e.g., polyoxyethylene (20) sorbitan monooleate, e.g., TWEEN For example, Super Refined TM 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)).

[0231] Any suitable concentration of surfactant can be used. The concentration of the surfactant in the pharmaceutical composition can be selected based on the expected ratio of surfactant to therapeutic protein and the concentration of the therapeutic protein. In some embodiments of any pharmaceutical composition described herein, the concentration of the surfactant (e.g., PS20 or P188) is from about 0.001% (w / v) to about 2% (w / v), for example, 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%, about 0.08%, about 0.09%, about 10.10%, about 15.15%, about 16.16%, about 17.17%, about 18.18%, about 19.19%, about 20.20%, about 21.19%, about 22.20%, about 23.19%, about 24.20%, about 25.21%, about 26.22%, about 27.23%, about 28.24%, about 29.25%, about 30.26%, about 31.27%, about 32.27%, about 33.28%, about 34.29%, about 35.30%, about 36.31%, about 37.32%, about 38.33%, about 39.34%, about 40.35%, about 41.36%, about 42.37%, about 43.38%, about 44.39%, about 45.40%, about 46.41%, about 47.39%, about 48.41%, about 49.5 8%, 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 certain embodiments, the surfactant is P188 and the concentration of P188 is about 0.08% (w / v).

[0232] Any pharmaceutical composition described herein may include a stabilizer. Any suitable stabilizer may be used. For example, in some embodiments, the stabilizer is thiosorbitan, 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 specific embodiments, the amino acid is methionine.

[0233] Any suitable concentration of the stabilizer (e.g., methionine) can be used. For example, in some embodiments of any of the aforementioned pharmaceutical compositions, the concentration of the stabilizer (e.g., methionine) is from about 0.01 mM to about 50 mM, e.g., 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, about 50 mM, about 47mM, about 48mM, about 49mM, or about 50mM. 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 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 M to about 40mM, about 9mM to about 40mM, about 10mM to about 40mM, about 15mM to about 40mM, about 20mM to about 40mM, about 25mM to about 40mM, about 30mM to about 40mM, about 1mM to about 30mM, about 2mM to about 30mM, about 3mM to about 30mM, about 4mM to about 30mM, about 5mM to about 30mM, about 6mM to about 3 ... M 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, about 15mM to about 30mM, about 20mM to about 30mM, about 25mM to about 30mM,about 1mM to about 20mM, about 2mM to about 20mM, about 3mM to about 20mM, about 4mM to about 20mM, about 5mM to about 20mM, about 6mM to about 20mM, about 7mM to about 20mM, about 8mM to about 20mM, about 9mM to about 20mM, about 10mM to about 20mM, about 11mM to about 20mM, about 12mM to about 20mM, about 13mM to about 20mM, about 14mM to about 20mM, about 15mM to about 20mM, about 1mM to about 15mM, about 2mM to about 15mM, about 3mM to about 15mM, about 4mM to about 20mM, about 5mM to about 20mM, about 6mM to about 15mM M, about 7mM to about 15mM, about 8mM to about 15mM, about 9mM to about 15mM, about 10mM to about 15mM, about 11mM to about 15mM, about 12mM to about 15mM, about 13mM to about 15mM, about 14mM to about 15mM, about 1mM to about 10mM, about 2mM to about 10mM, about 3mM to about 10mM, about 4mM to about 10mM, about 5mM to about 10mM, about 6mM to about 10mM, about 7mM to about 10mM, about 8mM to about 10mM, about 9mM to about 10mM, about 1mM to about 5mM, about 2mM to about 5mM, about 3mM to about 5mM, or about 4mM to about 5mM.

[0234] In some embodiments, the concentration of methionine is 2.5 mM to 20 mM (e.g., 2.5 mM to 5 mM, 5 mM to 7.5 mM, 7.5 mM to 10 mM, 10 mM to 12.5 mM, 12.5 mM to 15 mM, 15 mM to 17.5 mM, or 17.5 mM to 20 mM, e.g., 3 mM to 18 mM, 4 mM to 16 mM, 5 mM to 14 mM, or 8 mM to 12 mM, e.g., about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM). In certain embodiments, the concentration of methionine is about 10 mM.

[0235] In pharmaceutical compositions wherein the therapeutic protein contains an Fc region (e.g., an antibody, e.g., a bispecific antibody, e.g., a TDB, e.g., an anti-CD20 / anti-CD3 antibody, e.g., mosunimotuzumab), oxidation of the methionine at position 257 of the Fc region is less than about 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 specific embodiments, oxidation of the methionine at position 257 of the Fc region is no greater than about 6% over two weeks at 40° C.

[0236] Any of the foregoing compositions (e.g., pharmaceutical compositions) may further include a buffer. Any suitable buffer may be used. In some embodiments, the buffer is histidine, acetate, phosphate, succinate, or a combination thereof. In some embodiments, the histidine is histidine acetate. Alternative buffers include sodium dihydrogen phosphate, disodium hydrogen phosphate, trisodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, tripotassium phosphate, or a mixture thereof.

[0237] In some cases, the concentration of the buffer (e.g., histidine, such as histidine acetate) is 5 mM to 20 mM. For example, the buffer can be 5 mM to 10 mM, 10 mM to 15 mM, or 15 mM to 20 mM, e.g., 6 mM to 18 mM, 7 mM to 16 mM, 8 mM to 15 mM, 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 certain instances, the concentration of the buffer (e.g., histidine, such as histidine acetate) can be, for example, 8 mM to 12 mM, for example, about 8 mM, about 9 mM, about 10 mM, about 11 mM, or about 12 mM. In some embodiments, the concentration of the buffer (e.g., histidine, such as histidine acetate or histidine HCl, such as L-histidine acetate or L-histidine HCl) is about 10 mM or 20 mM. In a specific embodiment, the buffer is histidine HCl at a concentration of about 20 mM. In another specific embodiment, the buffer is histidine acetate at a concentration of about 20 mM. In yet another specific embodiment, the buffer is histidine acetate at a concentration of about 10 mM.

[0238] In some embodiments, the pharmaceutical composition includes a tonicity agent, such as a carbohydrate (e.g., sucrose, glucose, dextrose, glycerol, glycerol, mannitol, and trehalose), an amino acid, or a salt (e.g., sodium chloride and potassium chloride). In embodiments where the tonicity agent is a sugar, the sugar can be, for example, sucrose, glucose, glycerol, or trehalose. In specific embodiments, the sugar is sucrose. The concentration of the tonicity agent (e.g., a sugar such as sucrose) can be from about 100 mM to about 500 mM. For example, the concentration of the tonicity agent (e.g., a sugar, such as sucrose) can be, for example, 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, or 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.

[0239] In certain embodiments, the pharmaceutical composition further comprises an antioxidant. In certain embodiments, the antioxidant is N-acetyl-DL-tryptophan. In certain embodiments, the concentration of N-acetyl-DL-tryptophan is 0.1mM to 0.5mM (e.g., about 0.1mM, about 0.15mM, about 0.2mM, about 0.25mM, about 0.3mM, about 0.35mM, about 0.4mM, about 0.45mM or about 0.5mM). In certain embodiments, the concentration of N-acetyl-DL-tryptophan is about 0.3mM.

[0240] The pH of the pharmaceutical composition can be any suitable pH. In some embodiments, the pharmaceutical composition has a pH in the range of 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, about 5.6, about 5.7, about 5.8, about 5.9, about 5.10, about 5.11, about 5.12, about 5.13, about 5.14, about 5.15, about 5.16, about 5.17, about 5.18, about 5.19, about 5.20, about 5.21, about 5.22, about 5.23, about 5.24, about 5.25, about 5.26, about 5.27, about 5.28, about 5.29, about 5.30, about 5.31, about 5.32, about 5.33, about 5.34, about 5.35, about 5.36, about 5.37, about 5.38, about 5.39, about 5.40, about 5.41, about 5.42, about 5.43, about 5.44, about 5.45, about 5.46, about 5.47, about 5.48, about 5.49, about .4, 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 pH of the pharmaceutical composition is 5.1 to 6.1. In a specific embodiment, the pH of the pharmaceutical composition is about 5.8. In another specific embodiment, the pH of the pharmaceutical composition is about 5.5.

[0241] In addition to the specific components described above, the pharmaceutical compositions of the present disclosure may optionally be prepared in the form of lyophilized formulations or aqueous solutions by mixing the therapeutic protein of the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutical carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium 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; proteins such as blood Albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides (e.g., glucose, mannose), disaccharides (e.g., sucrose, trehalose) or polysaccharides (e.g., dextrin) or sugar alcohols such as mannitol or sorbitan; chelating agents such as ethylenediaminetetraacetic acid (EDTA); salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0242] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0243] In some embodiments, the pharmaceutical composition is in unit dosage form (e.g., a liquid preparation for infusion, a liquid preparation for injection, or a liquid preparation for dilution). In specific embodiments, the pharmaceutical composition is a liquid preparation for dilution. In specific embodiments, the liquid preparation 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 between 20 ml and 40 ml (e.g., between 20 ml and 30 ml, between 30 ml and 40 ml, between 20 ml and 35 ml, between 25 ml and 40 ml, between 25 ml and 35 ml, or between 28 ml and 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 specific 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 between 10 ml and 20 ml (e.g., between 10 ml and 15 ml, between 15 ml and 20 ml, between 13 ml and 20 ml, between 10 ml and 17 ml, between 13 ml and 17 ml, or between 14 ml and 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 specific embodiments, the volume of the liquid formulation for dilution is about 15 ml.

[0244] In another specific embodiment, the liquid formulation for dilution is supplied 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 supplied in a container having a volume of about 2.5 ml. In some embodiments, the volume of the liquid formulation for dilution is between 0.2 ml and 2 ml (e.g., between 0.2 ml and 1.5 ml, between 0.5 ml and 2 ml, between 0.5 ml and 1 ml, or between 0.8 ml and 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 specific embodiments, the volume of the liquid formulation for dilution is about 0.5 ml, about 0.9 ml, or about 1 ml.

[0245] In another specific embodiment, the liquid preparation for dilution is supplied in the container with the volume of about 15ml (for example, about 10ml, about 11ml, about 12ml, about 13ml, about 14ml, about 15ml, about 16ml, about 17ml, about 18ml, about 19ml or about 20ml).In certain embodiments, the liquid preparation for dilution is supplied in the container with the volume of about 10ml.In certain embodiments, the volume for the liquid preparation of dilution is between 4ml to 12ml (for example, between 4ml to 8ml, between 8ml to 12ml, between 4ml to 10ml, between 6ml to 12ml, between 6ml to 10ml or between 7ml to 9ml; For example, about 4ml, about 5ml, about 6ml, about 7ml, about 8ml, about 9ml, about 10ml, about 11ml or about 12ml).In a specific embodiment, the volume for the liquid preparation of dilution is about 8ml.

[0246] In some embodiments, the liquid preparation is used to be diluted with a diluent. In some embodiments, the liquid preparation is used to be diluted with a saline solution. In some embodiments, the liquid preparation is used to be diluted with a normal saline solution. In some embodiments, the normal saline solution comprises sodium chloride (NaCl). In some embodiments, the normal saline solution comprises between 0.1% and 1.5% (e.g., between 0.1% and 1.2%, between 0.3% and 1.5%, between 0.4% and 0.5%, between 0.3% and 1%, between 0.8% and 1%, between 0.85% and 0.95%; for example, 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) of NaCl).

[0247] In some embodiments, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., a TDB or TCB; e.g., mosulatuzumab, gefizumab, sivusituzumab, or rumotuzumab) after dilution with normal saline solution is from 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, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 6 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 particular embodiments, the concentration of the therapeutic protein (e.g., an antibody; e.g., a bispecific antibody; e.g., a TDB or TCB; e.g., mosulatuzumab, gefuzumab, silvosetumab, or rumotuzumab) after dilution with normal saline solution 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, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TDB; e.g., mosunituzumab) after dilution with saline solution 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, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-CD20 / anti-CD3 bispecific antibody; anti-CD20 / anti-CD3 TCB; e.g., gefiruzumab) after dilution with saline solution is about 0.1 mg / ml or about 0.6 mg / ml.In certain embodiments, the concentration of the therapeutic antibody (e.g., antibody; bispecific antibody; anti-FcRH5 / anti-CD3 bispecific antibody; anti-FcRH5 / anti-CD3 TDB; e.g., silvostomumab) after dilution with normal saline solution is about 0.003 mg / ml, 0.03 mg / ml, or 0.3 mg / ml.

[0248] The formulations described herein may also contain more than one active ingredient that is necessary for the specific indication being treated, preferably with active ingredients that have 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 inhibitors and / or anti-hormonal agents, such as those described above). Such active ingredients are suitably present in an amount effective for the intended purpose.

[0249] The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980).

[0250] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0251] Preparations for in vivo administration are generally sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

[0252] 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.

[0253] In some embodiments, the shelf life is between about 1 month and 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) when stored at 5°C ± 3°C and protected from light. In some embodiments, the shelf life when stored at 5°C ± 3°C and protected from light is between about 1 month and about 72 months, between about 1 month and about 70 months, between about 1 month and about 65 months, between about 1 month and about 60 months, between about 1 month and about 55 months, between about 1 month and about 50 months, between about 1 month and about 48 months, between about 1 month and about 45 months, between about 1 month and about 40 months, between about 1 month and about 35 months, between about 1 month and about 30 months, between about 1 month and about 25 months, between about 1 month and about 24 months, between about 1 month and about 20 months, between about 1 month and about 25 months. between about 1 month and about 18 months, between about 1 month and about 15 months, between about 1 month and about 12 months, between about 1 month and about 9 months, between about 1 month and about 6 months, between about 1 month and about 3 months, between about 5 months and about 72 months, between about 5 months and about 70 months, between about 5 months and about 65 months, between about 5 months and about 60 months, between about 5 months and about 55 months, between about 5 months and about 50 months, between about 5 months and about 48 months, between about 5 months and about 45 months, between about 5 months and about 40 months, between about 5 months and about 35 months, between about 5 months and about 30 months , between about 5 months and about 25 months, between about 5 months and about 24 months, between about 5 months and about 20 months, between about 5 months and about 18 months, between about 5 months and about 15 months, between about 5 months and about 12 months, between about 5 months and about 9 months, between about 5 months and about 6 months, between about 10 months and about 72 months, between about 10 months and about 70 months, between about 10 months and about 65 months, between about 10 months and about 60 months, between about 10 months and about 55 months, between about 10 months and about 50 months, between about 10 months and about 48 months, between about 10 months and about 45 months between about 10 months and about 40 months, between about 10 months and about 35 months, between about 10 months and about 30 months, between about 10 months and about 25 months, between about 10 months and about 24 months, between about 10 months and about 20 months, between about 10 months and about 18 months, between about 10 months and about 15 months, between about 10 months and about 12 months, between about 12 months and about 72 months, between about 12 months and about 70 months, between about 12 months and about 65 months, between about 12 months and about 60 months, between about 12 months and about 55 months, between about 12 months and about 50 months,between about 12 months and about 48 months, between about 12 months and about 45 months, between about 12 months and about 40 months, between about 12 months and about 35 months, between about 12 months and about 30 months, between about 12 months and about 25 months, between about 12 months and about 24 months, between about 12 months and about 20 months, between about 12 months and about 18 months, between about 12 months and about 15 months, between about 18 months and about 72 months, between about 18 months and about 70 months, between about 18 months and about 65 months, between about 18 months and about 60 months, between about 18 months and about 55 months, between about 18 months and about 50 months, between about 18 months and about 48 months, between about 18 months and about 45 months. months, between about 18 months and about 40 months, between about 18 months and about 35 months, between about 18 months and about 30 months, between about 18 months and about 25 months, between about 18 months and about 24 months, between about 18 months and about 20 months, between about 24 months and about 72 months, between about 24 months and about 70 months, between about 24 months and about 65 months, between about 24 months and about 60 months, between about 24 months and about 55 months, between about 24 months and about 50 months, between about 24 months and about 48 months, between about 24 months and about 45 months, between about 24 months and about 40 months, between about 24 months and about 35 months, between about 24 months and about 30 months, between about 24 months and about between about 25 months, between about 30 months and about 72 months, between about 30 months and about 70 months, between about 30 months and about 65 months, between about 30 months and about 60 months, between about 30 months and about 55 months, between about 30 months and about 50 months, between about 30 months and about 48 months, between about 30 months and about 45 months, between about 30 months and about 40 months, between about 30 months and about 35 months, between about 30 months and about 36 months, between about 36 months and about 72 months, between about 36 months and about 70 months, between about 36 months and about 65 months, between about 36 months and about 60 months, between about 36 months and about 55 months, between about 36 months and about 50 months, between about 36 months and about 36 months, months and about 48 months, between about 36 months and about 45 months, between about 36 months and about 40 months, between about 40 months and about 72 months, between about 40 months and about 70 months, between about 40 months and about 65 months, between about 40 months and about 60 months, between about 40 months and about 55 months, between about 40 months and about 50 months, between about 40 months and about 48 months, between about 40 months and about 45 months, between about 42 months and about 72 months, between about 42 months and about 70 months, between about 42 months and about 65 months, between about 42 months and about 60 months, between about 42 months and about 55 months, between about 42 months and about 50 months, between about 42 months and about 48 months,between about 42 months and about 45 months, between about 46 months and about 72 months, between about 46 months and about 70 months, between about 46 months and about 65 months, between about 46 months and about 60 months, between about 46 months and about 55 months, between about 46 months and about 50 months, between about 46 months and about 48 months, between about 48 months and about 72 months, between about 48 months and about 70 months, between about 48 months and about 65 months, between about 48 months and about 60 months, between about 48 months and about 55 months, Between about 48 months and about 50 months, between about 50 months and about 72 months, between about 50 months and about 70 months, between about 50 months and about 65 months, between about 50 months and about 60 months, between about 50 months and about 55 months, between about 55 months and about 72 months, between about 55 months and about 70 months, between about 55 months and about 65 months, between about 55 months and about 60 months, between about 60 months and about 72 months, between about 60 months and about 70 months, or between about 60 months and about 65 months.

[0254] Stable pharmaceutical compositions can include, for example, no more than 1,000 particles per ml having a diameter of ≥2 μm. For example, the pharmaceutical composition can have 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 (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). In some embodiments, the carrier is water.

[0255] 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%, e.g., as assessed by SE-HPLC. In specific embodiments, the pharmaceutical composition has a purity of about 90% or greater as assessed by SE-HPLC or a purity of about 95% or greater as assessed by SE-HPLC. In some embodiments, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC for about 36 months or longer 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 purity as assessed by SE-HPLC for about 36 months or longer at about 5°C, e.g., 85% to 90%, 90% to 95%, or 95% to 100% purity as assessed by SE-HPLC for about 36 months or longer at about 5°C). In particular instances, the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC at about 5°C for about 42 months or longer (e.g., about 42 months, about 60 months, about 72 months, about 84 months, about 96 months or longer at about 5°C).

[0256] In some cases, the present disclosure provides a pharmaceutical composition having a purity of about 75% or greater as assessed by 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, 85% or greater, about 86% or greater as assessed by non-reducing CE-SDS assay). In some embodiments, the pharmaceutical composition has a purity of about 80% or more as assessed by non-reducing CE-SDS. For example, in some embodiments, the pharmaceutical composition has a purity of about 85% or more as assessed by non-reducing CE-SDS. In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by non-reducing CE-SDS assay for about 36 months or longer 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 purity, e.g., between 85% and 90%, between 90% and 95%, or between 95% and 100% purity as assessed by non-reducing CE-SDS assay). In some embodiments, the pharmaceutical composition has a purity of about 85% or greater as assessed by non-reducing CE-SDS assay for about 42 months or longer 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 purity, e.g., between 85% and 90%, between 90% and 95%, or between 95% and 100% purity as assessed by non-reducing CE-SDS assay).

[0257] III. Exemplary Therapeutic Proteins

[0258] The therapeutic proteins in the pharmaceutical compositions of the present disclosure include antibodies, such as bispecific antibodies, for example, T cell-dependent bispecific antibodies (TDBs) or T cell-engaging bispecific antibodies (TCBs). The antibody or bispecific antibody can bind to CD3 (e.g., an anti-CD3 antibody or an anti-CD3 bispecific antibody). The TDB can have an anti-CD3 arm and an anti-target arm, wherein the anti-CD3 arm has a binding domain that binds to CD3, and the anti-target arm binds to the target antigen. The anti-target arm of such a TDB can be, for example, an anti-CD20 arm, an anti-FcRH5 arm, or an anti-HER2 arm. The TCB can have at least one CD3 binding portion and at least one target antigen binding portion. In some embodiments, the TCB has one CD3 binding portion and two target antigen binding portions. The target antigen binding portion of the TCB can be, for example, CD20.

[0259] Anti-CD3 bispecific antibodies

[0260] Anti-CD3 bispecific antibodies that can be used in the methods of the present disclosure include any anti-CD3 bispecific antibodies described in PCT Publication No. WO 2015 / 095392 (e.g., TDB), 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 includes 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.

[0261] In some embodiments, the anti-CD3 antibody comprises at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequence of SEQ ID NO: 25 to SEQ ID NO: 28, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequence of SEQ ID NO: 29 to SEQ ID NO: 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 as in (a) and a VL domain as in (b). In certain 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. In certain instances, the CD3 binding domain may have the amino acid sequence of 40G5c or a derivative or clonal relative thereof. The structural and functional features of 40G5c are described, for example, in PCT Publication No. WO 2015 / 095392, which is incorporated herein by reference in its entirety.

[0262] 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 comprises: 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.

[0263] In some embodiments, the anti-CD3 antibody comprises at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequence of SEQ ID NO: 81 to SEQ ID NO: 84, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequence of SEQ ID NO: 85 to SEQ ID NO: 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 as in (a) and a VL domain as in (b). In certain 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. In certain instances, the CD3 binding domain may have the amino acid sequence of 38E4v1 or a derivative or clonal relative thereof. The structural and functional features of 38E4v1 are described, for example, in PCT Publication No. WO 2015 / 095392, which is incorporated herein by reference in its entirety.

[0264] Anti-CD3 bispecific antibodies that can be used in the methods of the present disclosure include any anti-CD3 T cell activating bispecific antigen binding molecule (e.g., TCB, e.g., 2+1 TCB) described in U.S. Patent No. 9,914,776, which is incorporated herein by reference in its entirety. 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; and 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 as in (a) and a VL domain as in (b). In certain embodiments, 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.

[0265] In certain embodiments, the CD3 binding domain binds to an epitope within a fragment of CD3 (e.g., human CD3 epsilon) consisting of amino acids 1 to 26 or 1 to 27 of human CD3 epsilon.

[0266] The CD3 binding domain of any of the above antibodies (e.g., TDB or TCB) can bind to a specific CD3 epitope. For example, the CD3 binding domain can contact the 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 certain embodiments, the antibody binds to an epitope consisting of one, two, three, four or five 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 the amino acids of human CD3ε at a distance of about 3.5 angstroms or less. In certain embodiments, the antibody binds to an epitope consisting of one, two, three, four or five 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 consisting of amino acids of human CD3ε (selected from Gln1, Asp2, Asn4, Glu6 and Met7). In a specific embodiment, the CD3 binding domain binds to an epitope that specifically includes Glu 6. In certain other embodiments, the antibody does not bind to an epitope that includes amino acid Glu 5 of human CD3 epsilon. In certain other embodiments, the antibody does not bind to an epitope that includes amino acids Gly 3 and Glu 5 of human CD3 epsilon.

[0267] Anti-CD3 epitopes can be determined by binding an anti-CD3 antibody to a peptide fragment of the epitope. Alternatively, anti-CD3 epitopes can be determined by alanine scanning mutagenesis. In certain instances, a reduction in binding of an anti-CD3 antibody to a mutated CD3 by about 20%, about 30%, about 50%, about 80%, or more indicates that the amino acid residue of CD3 that was mutated in the alanine scanning mutagenesis assay is an epitope residue for the anti-CD3 antibody. Alternatively, anti-CD3 epitopes can be determined by mass spectrometry. In some embodiments, the epitope is determined by crystallography.

[0268] In some embodiments, amino acids Q1 to M7 of CD3 are used to determine epitopes determined by crystallography.

[0269] In some embodiments, the epitope determined by crystallography can be prepared by combining an anti-CD3 antibody Fab dissolved in 0.15M NaCl, 25mM tris (pH 7.5) at 10 mg / ml with a 2-fold molar excess (1 mg) of CD3ε peptide and initially screening a sparse matrix of the precipitate in a sitting drop vapor diffusion format. Optimized crystals can be grown from a 1:1 mixture of a reservoir solution containing 70% v / v methyl-pentanediol and 0.1M HEPES buffer at pH 7.5. The reservoir can be used as a cryoprotectant. The crystals can be transferred to cryogenic temperatures by sudden immersion in liquid nitrogen. Diffraction data for the crystals can be collected at the Advanced Photon Source beamline 22ID (Argonne National Laboratory; Lemont, IL) using a MAR300 CCD detector. The recorded diffractions 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, Vol. 276: Macromolecular Crystallography, Section A, pp. 307-326, 1997, CW Carter, Jr. and R. M. Sweet, eds., Academic Press (New York)).

[0270] 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 was for 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 I.N., 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). Based on the Fo-Fc map, the CD3ε peptide was built into the structure.The crystal structure can then be determined using the programs REFMAC5 (Murshudov GN, Vagin AA, Dodson EJ (1997) Acta Crystallogr D53:240-255) and PHENIX (D. Liebschner, PV Afonine, ML Baker, G. Bunkóczi, VB Chem, TI Croll, B. Hintze, L.-W. Hung, S. Jain, AJ McCoy, NW Moriarty, RD Oeffner, BK Poon, MG Prisant, RJ Read, JS Richardson, DC Richardson, MD Sammito, OV Sobolev, DH Stockwell, TC Terwilliger, AG Urzhumtsev, LL Videou, CJ Williams and PD Adams. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D53:240-255). Cryst. (2019). D75, 861-877), the structure was refined using the maximum likelihood objective function, anisotropic single B-factor refinement method and TLS (translation-vibration-helix-rotation) refinement method to achieve convergence.

[0271] In certain other embodiments, the pharmaceutical composition comprises an antibody comprising a paratope that binds to the same epitope as the anti-CD3 antibodies described above.

[0272] Anti-CD20 / anti-CD3 bispecific antibodies

[0273] In embodiments of the present disclosure where the therapeutic protein is an anti-CD20 / anti-CD3 bispecific antibody, the anti-CD20 / anti-CD3 bispecific antibody may incorporate any of the features described herein, alone or in combination.

[0274] Mosutuzumab

[0275] In some embodiments, an anti-CD20 / anti-CD3 bispecific antibody (eg, anti-CD20 / anti-CD3 TDB; eg, mosunituzumab) comprises an anti-CD20 arm and an anti-CD3 arm. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises: (1) an anti-CD20 arm having a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of GYTFTSYNMH (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence of VVYYSNSYWYFDV (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acid sequence of RASSSVSYMH (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence of APSNLAS (SEQ ID NO: 5); and (f) HVR-L3 comprising the amino acid sequence of QQWSFNPPT (SEQ ID NO: 6). NO:6); and (2) an anti-CD3 arm having a second binding domain comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of NYYIH (SEQ ID NO:9); (b) HVR-H2 comprising the amino acid sequence of WIYPGDGNTKYNEKFKG (SEQ ID NO:10); (c) HVR-H3 comprising the amino acid sequence of DSYSNYYFDY (SEQ ID NO:11); (d) HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:12); (e) HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:13); and (f) HVR-L3 comprising the amino acid sequence of TQSFILRT (SEQ ID NO:14).In some instances, mosulatuzumab comprises: (1) an anti-CD20 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NOs: 17 to 20, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NOs: 21 to 24, respectively; and (2) an anti-CD3 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NOs: 25 to 28, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NOs: 29 to 30, respectively. At least one (eg, 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequence of NO: 32. In some instances, mosulatuzumab comprises: (1) an anti-CD20 arm comprising a first binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO:7, or the amino acid sequence of SEQ ID NO:7; (b) a VL domain 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 the amino acid sequence of SEQ ID NO:8, or the amino acid sequence of SEQ ID NO:9. NO:8; or (c) a VH domain as in (a) and a VL domain as in (b); and (2) an anti-CD3 arm comprising a second binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO:15, or the amino acid sequence of SEQ ID NO:15; (b) a VL domain 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 the amino acid sequence of SEQ ID NO:16, or the amino acid sequence of SEQ ID NO:17. NO: the amino acid sequence of 16; or (c) the VH domain as in (a) and the VL domain as in (b).In some instances, mosulatuzumab 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.

[0276] In some instances, mosunituzumab 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 sequence and light chain sequence of SEQ ID NO: 33 and SEQ ID NO: 34, respectively; and (2) an anti-CD3 arm comprising the heavy chain and light chain sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0277] In some instances, mosulatuzumab comprises: (1) an anti-CD20 arm comprising a first binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO: 33, or the amino acid sequence of SEQ ID NO: 33; and (b) a light chain 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 the amino acid sequence of SEQ ID NO: 34, or the amino acid sequence of SEQ ID NO: 35. NO:34; or (c) a heavy chain as in (a) and a light chain as in (b); and (2) an anti-CD3 arm comprising a second binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO:35, or the amino acid sequence of SEQ ID NO:35; (b) a light chain 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 the amino acid sequence of SEQ ID NO:36, or the amino acid sequence of SEQ ID NO:36; or (c) a heavy chain as in (a) and a light chain as in (b). In some instances, mosulatuzumab 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.

[0278] The amino acid sequence of mosunituzumab is summarized in Table 1 below.

[0279] Table 1. Sequence ID of mosunituzumab

[0280]

[0281]

[0282] Mosunituzumab can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567.

[0283] Gefituzumab

[0284] In some other embodiments, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises: at least one antigen-binding domain that specifically binds to CD20, the antigen-binding domain 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: NO:42); and at least one antigen-binding domain that specifically binds to CD3, the antigen-binding domain 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 GTNCRAP (SEQ ID NO:49); and HVR-L3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO:50).

[0285] In some examples, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as gefuzumab) comprises: at least one antigen-binding domain that specifically binds to CD20, the antigen-binding domain comprising a VH domain 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 the amino acid sequence of SEQ ID NO: 43, or the amino acid sequence of SEQ ID NO: 43; and a VL domain 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 the amino acid sequence of SEQ ID NO: 44, or the amino acid sequence of SEQ ID NO: 45. NO:44; or (c) a VH domain as in (a) and a VL domain as in (b); and at least one antigen-binding domain that specifically binds to CD3, the antigen-binding domain comprising a VH domain 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 the amino acid sequence of SEQ ID NO:51, or the amino acid sequence of SEQ ID NO:51; and a VL domain 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 the amino acid sequence of SEQ ID NO:52, or the amino acid sequence of SEQ ID NO:52.

[0286] In one embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) is an antibody fragment, particularly a Fab molecule or a 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., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) is a crossover Fab molecule in which the variable domains or constant domains of the Fab heavy chain and the Fab light chain are exchanged (i.e., replaced with each other).

[0287] In one embodiment, the antigen-binding domain that specifically binds to CD20 of an anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) is an antibody fragment, particularly a Fab molecule or a 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., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) is a conventional Fab molecule.

[0288] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises at least one antigen binding domain that specifically binds to CD20 and 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, such as Gefituzumab) comprises a first antigen binding domain that specifically binds to CD3 and a second and third antigen binding domain that specifically binds 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 a conventional Fab molecule. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) further comprises an Fc domain. The anti-CD20 / anti-CD3 bispecific antibody may comprise modifications in the Fc region and / or in the antigen binding domain as described herein. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises an IgG1 Fc domain comprising amino acid substitutions L234A, L235A, and P329G (EU numbering).

[0289] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises: an antigen binding domain that specifically binds to CD3 fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; a first antigen binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc heavy chain of the antigen binding domain that specifically binds to CD3; and a second antigen binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0290] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises: (a) a first Fab molecule that specifically binds to CD3, in particular to CD3ε; and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other; (b) a second Fab and a third Fab molecule that specifically binds to CD20, wherein in the constant domain CL of the second Fab and the third Fab molecule, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted with lysine (K) or arginine (R), in particular arginine (R) (according to Kabat numbering), and wherein in the constant domain CH1 of the second Fab and the third Fab molecule, 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); and (c) an Fc domain composed of a first subunit and a second subunit capable of stably associating.

[0291] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0292] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (eg, an anti-CD20 / anti-CD3 TCB, such as Gefituzumab) is bivalent for CD20 and monovalent for CD3.

[0293] In one embodiment, the first Fab molecule (i.e., it binds CD3) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, the second Fab molecule (i.e., it binds CD20) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the heavy chain of the first Fab molecule, and the third Fab molecule (i.e., it binds CD20) is fused at the C-terminus of the Fab heavy chain to the N-terminus of another subunit of the Fc domain. In one embodiment, the first Fab molecule comprises: a VH domain that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 51; and a VL domain that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 52. In some embodiments, the first Fab molecule comprises: a heavy chain variable region sequence that comprises the amino acid sequence of SEQ ID NO: 51; and a light chain variable region that comprises the amino acid sequence of SEQ ID NO: 52.

[0294] In one embodiment, the second Fab molecule and the third Fab molecule each comprise a heavy chain variable region and a light chain variable region, the heavy chain variable region is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:43, and the light chain variable region is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:44.

[0295] In one embodiment, the second Fab molecule and the third Fab molecule each comprise a heavy chain variable region sequence of SEQ ID NO:43 and a light chain variable region sequence of SEQ ID NO:44.

[0296] In specific embodiments, the anti-CD20 / anti-CD3 bispecific antibody (e.g., an anti-CD20 / anti-CD3 TCB; e.g., Gefituzumab) 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 yet another specific embodiment, 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 another 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 of which comprises the amino acid sequence of SEQ ID NO: 56.

[0297] Specific anti-CD20 / anti-CD3 bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 and European Patent Application Nos. EP15188093 and EP16169160 (each herein incorporated by reference in its entirety).

[0298] The sequences of Gefituzumab are summarized in Table 2.

[0299] Table 2. Sequence ID of Gefituzumab

[0300]

[0301] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody of the pharmaceutical composition of the present disclosure is Gefituzumab.

[0302] Anti-FcRH5 / anti-CD3 bispecific antibody

[0303] In embodiments of the present disclosure where the therapeutic protein is an anti-FcRH5 / anti-CD30 bispecific antibody, the anti-FcRH5 / anti-CD3 bispecific antibody may incorporate any of the features described herein, alone or in combination.

[0304] Sivodizumab

[0305] In some embodiments, an anti-FcRH5 / anti-CD3 bispecific antibody (eg, an anti-FcRH5 / anti-CD3 TDB; eg, silvosumab) comprises an anti-FcRH5 arm and an anti-CD3 arm. In some embodiments, the anti-CD79b / anti-CD3 bispecific antibody comprises: (1) an anti-CD79b arm having a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of RFGVH (SEQ ID NO: 57); (b) HVR-H2 comprising the amino acid sequence of VIWRGGSTDYNAAFVS (SEQ ID NO: 58); (c) HVR-H3 comprising the amino acid sequence of HYYGSSDYALDN (SEQ ID NO: 59); (d) HVR-L1 comprising the amino acid sequence of KASQDVRNLVV (SEQ ID NO: 60); (e) HVR-L2 comprising the amino acid sequence of SGSYRYS (SEQ ID NO: 61); and (f) HVR-L3 comprising the amino acid sequence of QQHYSPPYT (SEQ ID NO: NO:62) amino acid sequence; 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 SYYIH (SEQ ID NO:65); (b) HVR-H2 comprising the amino acid sequence of WIYPENDNTKYNEKFKD (SEQ ID NO:66); (c) HVR-H3 comprising the amino acid sequence of DGYSRYYFDY (SEQ ID NO:67); (d) HVR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:68); (e) HVR-L2 comprising the amino acid sequence of WTSTRKS (SEQ ID NO:69); and (f) HVR-L3 comprising the amino acid sequence of KQSFILRT (SEQ ID NO:70).In some instances, silvositamab comprises: (1) an anti-FcRH5 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NO: 73 to SEQ ID NO: 76, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NO: 77 to SEQ ID NO: 80, respectively; and (2) an anti-CD3 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NO: 81 to SEQ ID NO: 84, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NO: 85 to SEQ ID NO: 86, respectively. NO: 88. In some instances, sivosetumab comprises: (1) an anti-FcRH5 arm comprising a first binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO: 63, or the amino acid sequence of SEQ ID NO: 63; (b) a VL domain 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 the amino acid sequence of SEQ ID NO: 64, or the amino acid sequence of SEQ ID NO: 65. NO:64; or (c) a VH domain as in (a) and a VL domain as in (b); and (2) an anti-CD3 arm comprising a second binding domain, the second binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO:71, or the amino acid sequence of SEQ ID NO:71; (b) a VL domain 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 the amino acid sequence of SEQ ID NO:72, or the amino acid sequence of SEQ ID NO:73. NO:72 amino acid sequence; or (c) the VH domain as in (a) and the VL domain as in (b).In some instances, silvositamab 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.

[0306] In some cases, silvostomomab is described in WHO Drug Information (International Nonproprietary Name), Recommended INN: List 84, Volume 34, Issue 3, 2020 publication (see page 701), has CAS Registry Number 1905409-39-3, and has (1) an anti-FcRH5 arm comprising the heavy chain sequence and light chain sequence of SEQ ID NO: 89 and SEQ ID NO: 90, respectively; and (2) an anti-CD3 arm comprising the heavy chain and light chain sequences of SEQ ID NO: 91 and SEQ ID NO: 92, respectively.

[0307] In some instances, silvositamab comprises: (1) an anti-FcRH5 arm comprising a first binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO: 89, or the amino acid sequence of SEQ ID NO: 89; and (b) a light chain 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 the amino acid sequence of SEQ ID NO: 90, or the amino acid sequence of SEQ ID NO: 91. NO:90; or (c) a heavy chain as in (a) and a light chain as in (b); and (2) an anti-CD3 arm comprising a second binding domain, the second binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO:91, or the amino acid sequence of SEQ ID NO:91; (b) a light chain 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 the amino acid sequence of SEQ ID NO:92, or the amino acid sequence of SEQ ID NO:92; or (c) a heavy chain as in (a) and a light chain as in (b). In some instances, silvositamab 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.

[0308] The amino acid sequence of silvositumomab is summarized in Table 3 below.

[0309] Table 3. Sequence ID of Sivoduzumab

[0310]

[0311] Sivoduzumab can be produced using recombinant methods and compositions, such as those described in US Pat. No. 4,816,567.

[0312] Anti-HER2 / anti-CD3 bispecific antibodies

[0313] In embodiments of the present disclosure where the therapeutic protein is an anti-HER2 / anti-CD3 bispecific antibody, the anti-HER2 / anti-CD3 bispecific antibody may incorporate any of the features described herein, alone or in combination.

[0314] Rumorumab

[0315] In some embodiments, the anti-HER2 / anti-CD3 bispecific antibody (eg, anti-HER2 / anti-CD3 TDB; eg, rumatuzumab) comprises an anti-HER2 arm and an anti-CD3 arm. In some embodiments, the anti-HER2 / anti-CD3 bispecific antibody comprises: (1) an anti-HER2 arm having a first binding domain comprising at least one, two, three, four, five or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of DTYIH (SEQ ID NO: 93); (b) HVR-H2 comprising the amino acid sequence of RIYPTNGYTRYADSVKG (SEQ ID NO: 94); (c) HVR-H3 comprising the amino acid sequence of WGGDGFYAMDY (SEQ ID NO: 95); (d) HVR-L1 comprising the amino acid sequence of RASQDVNTAVA (SEQ ID NO: 96); (e) HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 97); and (f) HVR-L3 comprising the amino acid sequence of QQHYTTPPT (SEQ ID NO: 98). NO:98) amino acid sequence; 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 instances, rufazumab comprises: (1) an anti-HER2 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NO: 101 to SEQ ID NO: 104, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NO: 105 to SEQ ID NO: 108, respectively; and (2) an anti-CD3 arm comprising at least one (e.g., 1, 2, 3, or 4) of the heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 of the sequences of SEQ ID NO: 117 to SEQ ID NO: 120, respectively, and / or at least one (e.g., 1, 2, 3, or 4) of the light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 of the sequences of SEQ ID NO: 121 to SEQ ID NO: NO: 124. In some instances, rumetuzumab comprises: (1) an anti-HER2 arm comprising a first binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO: 99, or the amino acid sequence of SEQ ID NO: 99; (b) a VL domain 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 the amino acid sequence of SEQ ID NO: 100, or the amino acid sequence of SEQ ID NO: 101. NO: 100; or (c) a VH domain as in (a) and a VL domain as in (b); and (2) an anti-CD3 arm comprising a second binding domain, the second binding domain comprising: (a) a VH domain 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 the amino acid sequence of SEQ ID NO: 115, or the amino acid sequence of SEQ ID NO: 115; (b) a VL domain 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 the amino acid sequence of SEQ ID NO: 116, or the amino acid sequence of SEQ ID NO: 117. NO:116 amino acid sequence; or (c) the VH domain as in (a) and the VL domain as in (b).In some instances, rufazumab 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.

[0316] In some instances, mosulatuzumab has International Nonproprietary Name (INN) Listing 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 heavy and light chain sequences of SEQ ID NO: 125 and SEQ ID NO: 126, respectively; and (2) an anti-CD3 arm comprising heavy and light chain sequences of SEQ ID NO: 128 and SEQ ID NO: 127, respectively.

[0317] In some instances, rufaxine comprises: (1) an anti-HER2 arm comprising a first binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO: 125, or the amino acid sequence of SEQ ID NO: 125; and (b) a light chain 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 the amino acid sequence of SEQ ID NO: 126, or the amino acid sequence of SEQ ID NO: 127. NO: 126; or (c) a heavy chain as in (a) and a light chain as in (b); and (2) an anti-CD3 arm comprising a second binding domain, the second binding domain comprising: (a) a heavy chain 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 the amino acid sequence of SEQ ID NO: 127, or the amino acid sequence of SEQ ID NO: 127; (b) a light chain 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 the amino acid sequence of SEQ ID NO: 128, or the amino acid sequence of SEQ ID NO: 128; or (c) a heavy chain as in (a) and a light chain as in (b). In some instances, rufazumab 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.

[0318] The amino acid sequence of rumizumab is summarized in Table 4 below.

[0319] Table 4. Sequence ID of rufazumab

[0320]

[0321] Rumorumab can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567.

[0322] IV. Antibody Properties, Modifications, and Characterization Methods

[0323] The pharmaceutical compositions provided herein may further include any of the antibodies described herein, wherein the antibody comprises a VH as in any embodiment provided above and a VL as in any embodiment provided above, wherein one or both of the constant domain sequences include a post-translational modification. Additionally or alternatively, the antibody according to any of the above embodiments (e.g., a bispecific antibody, such as TDB or TCB) may incorporate any of the features described below, alone or in combination.

[0324] 1. Antibody affinity

[0325] In certain embodiments, the antibodies of the pharmaceutical compositions and methods provided herein have, for example, a 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (K D ).

[0326] In one embodiment, K is measured by radiolabeled antigen binding assay (RIA). D In one embodiment, RIA is performed using a Fab form of the antibody of interest and its antigen. For example, by using a minimal concentration of ( 125 The solution binding affinity of Fab for antigen was measured by equilibrating Fab with 1) labeled antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, 5 μg / ml capture anti-Fab antibody (Cappel Labs) was used to coat the plate with 1) labeled antigen. Microplates (Thermo Scientific) were blocked overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125The antigen is mixed with a serial dilution of the Fab of interest (e.g., following 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; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and washed with 0.1% polysorbate 20 (PBS) in water. 20) Wash the plate eight times. When the plate has dried, add 150 μL / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM The plates were counted for tens of minutes on a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in the competitive binding assay.

[0327] According to another embodiment, using Surface plasmon resonance assay to measure K D For example, using -2000 or -3000( Inc., Piscataway, NJ) was used to perform the assay using an immobilized antigen CM5 chip at approximately 10 response units (RU) at 25° C. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, Inc.). Antigen was diluted to 5 μg / ml (about 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 the injection of antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected at 25°C at a flow rate of about 25 μl / min into a 1% PBS solution containing 0.05% polysorbate 20 ( 20) Surfactant (PBST) in PBS. Using a simple one-to-one Langmuir binding model ( Evaluation Software version 3.2), the association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by the surface plasmon resonance assay described above exceeds 10 6 M- 1 s- 1 , the association rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation wavelength = 295 nm; emission wavelength = 340 nm, bandpass 16 nm) of a 20 nM antigen-antibody (Fab form) in 25°C PBS (pH 7.2) in the presence of increasing concentrations of antigen, which can be measured using a spectrophotometer such as a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette. TM Measured by spectrophotometer (ThermoSpectronic).

[0328] 2. Antibody fragments

[0329] In certain embodiments, the therapeutic protein is an antibody fragment, such as 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 certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün in 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. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0330] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003).

[0331] 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, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0332] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0333] 3. Chimeric and humanized antibodies

[0334] 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 in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0335] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Humanized antibodies optionally also include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0336] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, 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"); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0337] 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 the consensus sequence of human antibodies of a particular subgroup 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)). 93)); 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 FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0338] 4. Knob-Mortar Antibody Engineering

[0339] Antibodies (e.g., bispecific antibodies, such as TDB or TCB) can be prepared as full-length antibodies or antibody fragments. Techniques for preparing bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein et al., Nature 305:537 (1983)); WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655 (1991)) and "knob-to-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). The "knob-to-hole" engineering of bispecific antibodies can be used to generate a first subunit of the Fc domain containing the knob (e.g., a subunit of the Fc domain of the first arm) and a second subunit of the Fc domain containing the hole (e.g., a subunit of the Fc domain of the second arm) to which the knob of the first arm can be bound. In one embodiment, the knob of the bispecific antibody can be on one subunit of the Fc domain (e.g., the Fc subunit of the anti-CD3 arm). Alternatively, the knob of the bispecific antibody of the present disclosure may be on another subunit of the Fc domain (e.g., the Fc subunit of the anti-target arm; e.g., the Fc subunit of the anti-CD20, anti-FcRH5, or anti-HER2 arm). In one embodiment, the hole of the bispecific antibody of the present disclosure may be on the anti-CD3 arm. Alternatively, the hole of the bispecific antibody of the present disclosure may be on the anti-target arm (e.g., anti-CD20, anti-FcRH5, or anti-HER2 arm). In some cases, the anti-CD20 / anti-CD3 bispecific antibody produced using the knob-hole technology may comprise 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 cases, at least one of one or more heavy chain constant domains is paired with another heavy chain constant domain. In some cases, each of the CH31 and CH32 domains comprises a projection or a cavity, and wherein the projection or cavity in the CH31 domain can be positioned in the cavity or projection in the CH32 domain respectively. In some cases, the CH31 and CH32 domains meet at the interface between the projection and the cavity. In some cases, each of the CH21 and CH22 domains comprises a projection or a cavity, and wherein the projection or cavity in the CH21 domain can be positioned in the cavity or projection in the CH22 domain respectively. In some cases, the CH21 and CH22 domains meet at the interface between the projection and the cavity.

[0340] Bispecific antibodies can also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, for example, WO 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108: 11187-11192 (2011)). Bispecific antibodies can also be prepared by the following methods: engineering electrostatic manipulation effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980; and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments segment (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., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0341] Bispecific antibodies or antibody fragments thereof may also include "dual-acting Fabs" or "DAFs" (see, e.g., U.S. Publication No. 2008 / 0069820; Bostrom et al., 2009, Science 323:1610-14), which contain antigen binding sites that bind to CD3 and a target antigen such as CD20, FcRH5, or HER2.

[0342] 5. Antibody variants

[0343] In some cases, it is contemplated that the amino acid sequence variants of the above-mentioned antibodies. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct has desired characteristics, for example, antigen binding.

[0344] (i) Substitution, insertion and deletion variants

[0345] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target site for substitution mutagenesis includes HVR and framework region (FR). Conservative substitutions are shown under the "preferred substitutions" heading in Table 5. More substantial changes are provided under the "exemplary substitutions" heading in Table 5, and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0346] Table 5. Exemplary and preferred amino acid substitutions

[0347]

[0348]

[0349] Amino acids can be grouped according to common side chain properties:

[0350] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0351] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0352] (3) Acidic: Asp, Glu;

[0353] (4) Basic: His, Lys, Arg;

[0354] (5) Residues that affect chain orientation: Gly, Pro;

[0355] (6) Aromatic: Trp, Tyr, Phe.

[0356] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0357] One type of substitution variant involves replacing one or more hypervariable region (HVR) residues of a parent antibody (e.g., a humanized or human antibody). Typically, relative to the parent antibody, one or more resulting variants selected for further study will have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0358] For example, HVR can be changed (e.g., substituted) to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons undergo high-frequency mutations during the somatic maturation process (see, e.g., Chowdhury, Methods Mol.Biol.207:179-196 (2008)) and / or residues in contact with the antigen, and the resulting variant VH or VL binding affinity is tested. Affinity maturation achieved by constructing and reselecting from a secondary library has been described by, for example, Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis genes). A secondary library is then created. The library is subsequently screened to identify any antibody variant with the desired affinity. Another approach to introduce diversity involves an HVR-directed approach in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0359] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antigen-binding ability of the antibody. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be outside the antigen-contacting residues of HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or comprises no more than one, two or three amino acid substitutions.

[0360] As described in Cunningham and Wells (1989) (Science, 244:1081-1085), a method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and the antigen are identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they possess desired properties.

[0361] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions to the N-terminus or C-terminus of an antibody that increases the serum half-life of an antibody (e.g., for ADEPT) or a polypeptide.

[0362] (ii) Glycosylation variants

[0363] In some cases, the antibodies of the pharmaceutical compositions provided herein have been modified to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0364] In some embodiments, the present invention relates to an antibody comprising an Fc region, wherein the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide. In some embodiments, the oligosaccharide is a di-antennary oligosaccharide.

[0365] In some cases, the antibody of the pharmaceutical composition has a carbohydrate structure that lacks fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry is determined by calculating the average amount of fucose in the Asn297 sugar chain, for example, as disclosed in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, eg, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 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 that lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Publication No. US2003 / 0157108, Presta, L; and PCT Publication No. WO 2004 / 056312, Adams et al., particularly in Example 11); and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0366] In view of the above, in some cases, antibodies useful in the present disclosure include aglycosylation site mutations. In some cases, aglycosylation site mutations reduce the effector function of the bispecific antibody. In some cases, aglycosylation mutations are a substitution mutation. In some cases, the bispecific antibody comprises a substitution mutation in the Fc region that reduces the effector function. In some cases, the substitution mutations are at amino acid residues N297, L234, L235, D265, and / or P329 (EU numbering). In some cases, the substitution mutations are selected from the group consisting of: N297G, N297A, L234A, L235A, D265A, and P329G. In some cases, the substitution mutations are at amino acid residue N297. In a preferred embodiment, the substitution mutation is N297A.

[0367] In other cases, antibody variants with bisected oligosaccharides are used according to the methods of the present disclosure, for example, wherein the bisected oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having 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 in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0368] (iii) Fc region variants

[0369] In some cases, antibody variants having one or more amino acid modifications introduced into the Fc region of an antibody (e.g., a bispecific antibody, such as TDB or TCB) (i.e., Fc region variants (see, e.g., US2012 / 0251531)) can be administered to a subject having cancer (e.g., a B-cell proliferative disorder) according to the methods of the present disclosure. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0370] In some cases, bispecific Fc region antibody variants have some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 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, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay ( Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can 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). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can 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)). FcRn binding and in vivo clearance / half-life assays 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)).

[0371] Antibodies with reduced effector function include those with substitutions of one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent Nos. 6,737,056 and 8,219,149 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine ( U.S. Patent Nos. 7,332,581 and 8,219,149 ).

[0372] In some cases, the proline at position 329 of the wild-type human Fc region of the antibody is substituted with glycine or arginine or an amino acid residue sufficient to disrupt the proline sandwich within the Fc / Fcγ receptor interface, which is formed between proline 329 of Fc and tryptophan residues Trp 87 and Trp 110 of FcγRIII (Sondermann et al. Nature. 406, 267-273 (2000)). In certain embodiments, the bispecific antibody comprises at least one additional amino acid substitution. In another embodiment, the additional amino acid substitution is S228P, E233P...

Claims

1. A pharmaceutical composition comprising a therapeutic protein, polysorbate 20 (PS20), methionine, a buffer, and a carrier, wherein the molar ratio of the PS20 to the therapeutic protein is 100 or less, the concentration of the PS20 is 0.01% to 0.12% weight / volume (w / v), the concentration of the methionine is 1 mM to 50 mM, and the concentration of the buffer is 5 mM to 20 mM.

2. The pharmaceutical composition of claim 1, wherein the concentration of the therapeutic protein is about 10 mg / ml or less.

3. The pharmaceutical composition of claim 1 or 2, wherein the concentration of the therapeutic protein is between about 0.1 mg / ml to about 10 mg / ml.

4. The pharmaceutical composition of claim 3, wherein the therapeutic concentration is about 3 mg / ml or about 1 mg / ml.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the molar ratio of the PS20 to the therapeutic protein is 45 to 100.

6. The pharmaceutical composition of claim 5, wherein the molar ratio of the PS20 to the therapeutic protein is about 48, about 71, or about 79.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition is formulated as a pharmaceutical product (DP).

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the methionine is about 2.5 mM to about 20 mM.

9. The pharmaceutical composition of claim 7, wherein the concentration of the methionine is about 10 mM.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the buffer is about 5 mM to about 25 mM.

11. The pharmaceutical composition of claim 10, wherein the concentration of the buffer is about 10 mM or about 20 mM.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the buffer is histidine, phosphate, succinate, acetate, or a combination thereof.

13. The pharmaceutical composition according to claim 12, wherein the buffer is histidine. The pharmaceutical composition according to claim 13 , wherein the histidine is histidine acetate or histidine HCl.

15. The pharmaceutical composition according to any one of claims 10 to 14, wherein the buffer is histidine acetate at a concentration of about 10 mM or about 20 mM.

16. The pharmaceutical composition according to any one of claims 10 to 14, wherein the buffer is histidine HCl at a concentration of about 20 mM.

17. The pharmaceutical composition according to any one of claims 1 to 16, further comprising a tonicity agent.

18. The pharmaceutical composition of claim 17, wherein the tonicity agent is a sugar, an amino acid or a salt.

19. The pharmaceutical composition of claim 18, wherein the tonicity agent is a sugar.

20. The pharmaceutical composition according to claim 19, wherein the sugar is sucrose, glucose, glycerol or trehalose.

21. The pharmaceutical composition of claim 20, wherein the sugar is sucrose.

22. The pharmaceutical composition of any one of claims 17 to 21, wherein the concentration of the tonicity agent is about 100 mM to about 500 mM.

23. The pharmaceutical composition of claim 22, wherein the concentration of the tonicity agent is about 200 mM to about 300 mM.

24. The pharmaceutical composition of claim 23, wherein the concentration of the tonicity agent is about 240 mM.

25. The pharmaceutical composition of any one of claims 1 to 24, wherein the pharmaceutical composition has a pH of about 4.5 to about 8.

26. The pharmaceutical composition of claim 25, wherein the pH of the pharmaceutical composition is about 5.1 to about 6.

1.

27. The pharmaceutical composition of claim 26, wherein the pH of the pharmaceutical composition is about 5.5 or about 5.

8.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein the pharmaceutical composition further comprises an antioxidant.

29. The pharmaceutical composition of claim 28, wherein the antioxidant is N-acetyl-DL-tryptophan.

30. The pharmaceutical composition of claim 29, wherein the concentration of N-acetyl-DL-tryptophan is between 0.1 mM and 0.5 mM.

31. The pharmaceutical composition of claim 30, wherein the concentration of N-acetyl-DL-tryptophan is about 0.3 mM.

32. The pharmaceutical composition of any one of claims 1 to 31, wherein the therapeutic protein is an antibody.

33. The pharmaceutical composition of claim 32, wherein the antibody is a bispecific antibody.

34. The pharmaceutical composition of claim 33, wherein the bispecific antibody comprises at least one antigen binding domain that specifically binds to CD3 and at least one antigen binding domain that specifically binds to a target antigen.

35. The pharmaceutical composition of claim 34, wherein the bispecific antibody has a methionine at position 257 (EU numbering) of the Fc region, and wherein, Oxidation of the methionine at position 257 of the Fc region was less than about 10% over two weeks at 40°C.

36. The pharmaceutical composition according to claim 35, wherein The oxidation of the methionine at position 257 of the Fc region did not exceed about 6% over two weeks at 40°C.

37. A pharmaceutical composition comprising a bispecific antibody, a surfactant, methionine, and a carrier, wherein the pharmaceutical composition has a pH of about 5.5 or 5.8, and wherein: (i) the bispecific antibody comprises at least one antigen binding domain that specifically binds to CD3 and at least one antigen binding domain that specifically binds to a target antigen, and wherein the concentration of the bispecific antibody is about 10 mg / ml or less, (ii) the concentration of the surfactant is from about 0.05% to about 0.12% w / v, and (iii) The concentration of methionine is about 10 mM.

38. The pharmaceutical composition of claim 37, wherein the molar ratio of the surfactant to the bispecific antibody is 100 or less.

39. The pharmaceutical composition according to claim 37 or 38, wherein the surfactant is PS20 or poloxamer 188 (P188).

40. The pharmaceutical composition of claim 39, wherein the surfactant is PS20 and the concentration of PS20 is about 0.05%, 0.06%, or about 0.12% w / v.

41. The pharmaceutical composition of claim 40, wherein the molar ratio of the PS20 to the bispecific antibody is about 45 to about 100.

42. The pharmaceutical composition of claim 41, wherein the molar ratio of the PS20 to the bispecific antibody is about 48, about 71, or about 79.

43. The pharmaceutical composition of claim 39, wherein the surfactant is P188 and the concentration of P188 is about 0.1% w / v.

44. The pharmaceutical composition of claim 43, wherein the molar ratio of the P188 to the bispecific antibody is from about 5 to about 25.

45. The pharmaceutical composition of claim 44, wherein the molar ratio of P188 to the bispecific antibody is about 17.

46. ​​The pharmaceutical composition of any one of claims 37 to 45, wherein the concentration of the bispecific antibody is between about 0.1 mg / ml to about 5 mg / ml.

47. The pharmaceutical composition of claim 46, wherein the concentration of the bispecific antibody is about 1 mg / ml or about 3 mg / ml.

48. The pharmaceutical composition according to any one of claims 37 to 47, wherein the pharmaceutical composition is formulated as a DP.

49. The pharmaceutical composition of any one of claims 37 to 48, wherein the pharmaceutical composition further comprises histidine at a concentration of about 10 mM or about 20 mM.

50. The pharmaceutical composition of claim 49, wherein the histidine is histidine acetate or histidine HCl.

51. The pharmaceutical composition of any one of claims 37 to 50, wherein the pharmaceutical composition further comprises sucrose at a concentration of about 240 mM.

52. A pharmaceutical composition according to any one of claims 37 to 51, wherein the carrier is water.

53. A pharmaceutical composition according to any one of claims 32 to 52, wherein the pharmaceutical composition further comprises an antioxidant.

54. The pharmaceutical composition of claim 53, wherein the antioxidant is N-acetyl-DL-tryptophan.

55. The pharmaceutical composition of claim 54, wherein the concentration of N-acetyl-DL-tryptophan is between 0.1 mM and 0.5 mM.

56. The pharmaceutical composition of claim 55, wherein the concentration of N-acetyl-DL-tryptophan is about 0.3 mM.

57. A pharmaceutical composition according to any one of claims 32 to 56, wherein the bispecific antibody comprises an anti-CD3 arm and an anti-target arm.

58. The pharmaceutical composition of claim 57, wherein the anti-target arm is an anti-FcRH5 arm.

59. The pharmaceutical composition of claim 58, wherein the anti-CD3 arm comprises a CD3 binding domain comprising: (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) HVR-L3 comprising the amino acid sequence of SEQ ID NO:

70.

60. The pharmaceutical composition of claim 59, 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 as in (a) and a VL domain as in (b).

61. The pharmaceutical composition of claim 60, 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.

62. The pharmaceutical composition of any one of claims 58 to 61, wherein the anti-FcRH5 arm comprises a FcRH5 binding domain comprising: (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) HVR-L3 comprising the amino acid sequence of SEQ ID NO:

62.

63. The pharmaceutical composition of claim 62, 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 as in (a) and a VL domain as in (b).

64. The pharmaceutical composition of claim 63, 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.

65. A pharmaceutical composition comprising a bispecific antibody, PS20, methionine, a buffer, and a carrier, wherein the molar ratio of PS20 to the bispecific antibody is about 100 or less, the concentration of PS20 is about 0.01% to about 0.12% w / v, the concentration of methionine is 1 mM to 50 mM, and the concentration of the buffer is 5 mM to 20 mM, wherein the bispecific antibody comprises an anti-CD3 arm and an anti-FcRH5 arm, and wherein: (a) the anti-CD3 arm comprises a CD3 binding domain, wherein the CD3 binding domain comprises: 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; and (b) the anti-FcRH5 arm comprises a FcRH5 binding domain, wherein the FcRH5 binding domain comprises: 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.

66. The pharmaceutical composition of claim 65, 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 as in (a) and a VL domain as in (b); And the FcRH5 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 63; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 64; or (c) a VH domain as in (a) and a VL domain as in (b).

67. The pharmaceutical composition of claim 66, wherein: (a) 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, and (b) 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.

68. The pharmaceutical composition of claim 57, wherein the anti-target arm is an anti-HER2 arm.

69. The pharmaceutical composition of claim 68, wherein the anti-CD3 arm comprises a CD3 binding domain comprising: (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.

70. The pharmaceutical composition of claim 69, 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 as in (a) and a VL domain as in (b).

71. The pharmaceutical composition of claim 70, 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.

72. The pharmaceutical composition of any one of claims 68 to 71, wherein the anti-HER2 arm comprises a HER2 binding domain 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.

73. The pharmaceutical composition of claim 72, 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 as in (a) and a VL domain as in (b).

74. The pharmaceutical composition of claim 73, 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.

75. A pharmaceutical composition comprising a bispecific antibody, PS20, methionine, a buffer, and a carrier, wherein the molar ratio of PS20 to the bispecific antibody is about 100 or less, the concentration of PS20 is about 0.01% to about 0.12% w / v, the concentration of methionine is 1 mM to 50 mM, and the concentration of the buffer is 5 mM to 20 mM, wherein the bispecific antibody comprises an anti-CD3 arm and an anti-HER2 arm, and wherein: (a) the anti-CD3 arm comprises a CD3 binding domain, wherein the CD3 binding domain comprises: 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; and (b) the anti-HER2 arm comprises a HER2 binding domain, wherein the HER2 binding domain comprises: 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.

76. The pharmaceutical composition of claim 75, 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 as in (a) and a VL domain as in (b); And the HER2 binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 99; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 100; or (c) a VH domain as in (a) and a VL domain as in (b).

77. The pharmaceutical composition of claim 76, wherein: (a) 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, and (b) 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.

78. The pharmaceutical composition of claim 32, wherein the antibody is an IgG antibody, or the pharmaceutical composition of any one of claims 33, 34, and 57 to 77, wherein the bispecific antibody is an IgG antibody.

79. The pharmaceutical composition of claim 78, wherein the IgG antibody is an IgG1 antibody.

80. The pharmaceutical composition of claim 78 or 79, wherein the bispecific antibody comprises one or more substitution mutations in the Fc region.

81. The pharmaceutical composition of claim 80, wherein the bispecific antibody comprises a deglycosylation site mutation.

82. The pharmaceutical composition of claim 81, wherein the deglycosylation site mutation is a substitution mutation.

83. The pharmaceutical composition of claim 80, wherein the one or more substitution mutations reduce the effector function of the bispecific antibody.

84. The pharmaceutical composition of claim 80, 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).

85. The pharmaceutical composition of claim 84, wherein the substitution mutation is at least one selected from the group consisting of N297A, N297G, L234A, L235A, D265A and P329G (EU numbering).

86. The pharmaceutical composition of claim 80, wherein the one or more substitution mutations in the Fc region comprise one or more knob-to-hole mutations.

87. A pharmaceutical composition according to any one of claims 78 to 86, wherein the anti-target arm comprises T366W and N297G substitution mutations, and the anti-CD3 arm comprises T366S, L368A, Y407V and N297G substitution mutations.

88. The pharmaceutical composition according to any one of claims 58 to 67 and 78 to 87, wherein the bispecific antibody is sivostatin.

89. The pharmaceutical composition of any one of claims 68 to 87, wherein the bispecific antibody is rumoxetine.

90. A pharmaceutical composition according to any one of claims 32 to 89, wherein the at least one antigen binding domain that specifically binds to CD3 binds to a human CD3 polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide.

91. The pharmaceutical composition of claim 90, wherein the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3ε polypeptide or a cyno CD3ε polypeptide, respectively.

92. The pharmaceutical composition of claim 90, wherein the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3γ polypeptide or a cyno CD3γ polypeptide, respectively.

93. A pharmaceutical composition according to any one of claims 32 to 92, wherein the bispecific antibody is monoclonal.

94. A pharmaceutical composition according to any one of claims 32 to 93, wherein the bispecific antibody is human, humanized or chimeric.

95. The pharmaceutical composition of any one of claims 1 to 94, wherein the pharmaceutical composition is in unit dosage form.

96. The pharmaceutical composition of claim 95, wherein the unit dosage form is a liquid preparation for dilution.

97. The pharmaceutical composition of claim 96, wherein the liquid formulation for dilution is supplied in a container having a volume of about 50 ml.

98. The pharmaceutical composition of claim 96, wherein the liquid formulation for dilution is supplied in a container having a volume of about 15 ml.

99. The pharmaceutical composition of claim 96, wherein the liquid formulation for dilution is supplied in a container having a volume of about 2 ml.

100. The pharmaceutical composition of claim 96, wherein the liquid formulation for dilution is supplied in a container having a volume of about 1 ml.

101. The pharmaceutical composition of claim 96 or 97, wherein the volume of the liquid formulation for dilution is about 15 ml or about 30 ml.

102. The pharmaceutical composition of claim 96 or 98, wherein the volume of the liquid formulation for dilution is about 8 ml or about 15 mL.

103. The pharmaceutical composition of claim 96 or 99, wherein the volume of the liquid formulation for dilution is about 0.5 ml or about 0.9 ml.

104. The pharmaceutical composition of claim 96 or 100, wherein the volume of the liquid formulation for dilution is about 0.5 ml or about 0.9 ml.

105. The pharmaceutical composition of any one of claims 96 to 104, wherein the liquid formulation is for dilution with a physiological saline solution comprising 0.45% or 0.9% (w / v) NaCl.

106. The pharmaceutical composition of any one of claims 1 to 105, wherein the pharmaceutical composition comprises no more than 1,000 particles having a diameter ≥ 2 μm per ml as detected by high accuracy liquid particle counting (HIAC).

107. The pharmaceutical composition of any one of claims 1 to 106, wherein the pharmaceutical composition has a shelf life of at least 36 months when stored at 5°C ± 3°C and protected from light.

108. The pharmaceutical composition of any one of claims 1 to 107, wherein the pharmaceutical composition is stable to one or more freeze-thaw cycles.

109. The pharmaceutical composition of claim 108, wherein the pharmaceutical composition is stable to three or more freeze-thaw cycles.

110. The pharmaceutical composition of any one of claims 1 to 109, wherein the pharmaceutical composition is stable at about 25°C for about two weeks or longer.

111. The pharmaceutical composition of claim 110, wherein the pharmaceutical composition is stable at about 25°C for about four weeks or longer.

112. The pharmaceutical composition of any one of claims 1 to 111, wherein the pharmaceutical composition is stable at -20°C for about 48 months or longer.

113. The pharmaceutical composition of any one of claims 108 to 112, wherein stability is assessed by size exclusion high performance liquid chromatography (SE-HPLC).

114. The pharmaceutical composition of claim 113, wherein the pharmaceutical composition is determined to be stable if the pharmaceutical composition maintains a purity as measured by SE-HPLC with a variation of less than 5%.

115. The pharmaceutical composition of any one of claims 108 to 112, wherein stability is assessed by non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) assay.

116. The pharmaceutical composition of claim 115, wherein the pharmaceutical composition is determined to be stable if it maintains a purity as measured by a non-reducing CE-SDS assay with a variation of less than 5%.

117. The pharmaceutical composition of claim 115 or 116, wherein the non-reduced CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

118. The pharmaceutical composition of any one of claims 1 to 117, wherein the pharmaceutical composition has a purity of about 85% or greater as assessed by SE-HPLC.

119. The pharmaceutical composition of claim 118, wherein the pharmaceutical composition has a purity of about 90% or greater as assessed by SE-HPLC.

120. The pharmaceutical composition of claim 119, wherein the pharmaceutical composition has a purity of about 95% or greater as assessed by SE-HPLC.

121. The pharmaceutical composition of any one of claims 118 to 120, wherein the purity of the pharmaceutical composition as assessed by SE-HPLC remains about the same at about 5°C for about 36 months or longer.

122. The pharmaceutical composition of claim 121, wherein the purity of the pharmaceutical composition as assessed by SE-HPLC remains about the same at about 5°C for about 42 months or longer.

123. The pharmaceutical composition of claim 122, wherein the purity of the pharmaceutical composition as assessed by SE-HPLC remains approximately the same at about 5°C for about 64 months or longer.

124. The pharmaceutical composition of any one of claims 1 to 117, wherein the pharmaceutical composition has a purity of about 75% or greater as assessed by non-reducing CE-SDS assay.

125. The pharmaceutical composition of claim 124, wherein the pharmaceutical composition has a purity of about 80% or greater as assessed by non-reducing CE-SDS assay.

126. The pharmaceutical composition of claim 125, wherein the pharmaceutical composition has a purity of about 85% or greater as assessed by non-reducing CE-SDS assay.

127. The pharmaceutical composition of any one of claims 124 to 126, wherein the purity of the pharmaceutical composition as assessed by non-reducing CE-SDS assay is maintained at about 5°C for about 36 months or longer.

128. The pharmaceutical composition of claim 127, wherein the purity of the pharmaceutical composition as assessed by non-reducing CE-SDS assay is maintained at about 5°C for about 42 months or longer.

129. The pharmaceutical composition of any one of claims 124 to 128, wherein the non-reduced CE-SDS assay is a microchip CE-SDS (mCE-SDS) assay.

130. The pharmaceutical composition of any one of claims 1 to 129, wherein the pharmaceutical composition is formulated for intravenous administration.

131. The pharmaceutical composition of any one of claims 1 to 130, wherein the pharmaceutical composition is preservative-free.

132. The pharmaceutical composition according to any one of claims 1 to 131, wherein the pharmaceutical composition is formulated for administration by infusion after dilution with a physiological saline solution comprising 0.45% or 0.9% NaCl.

133. A pharmaceutical composition according to any one of claims 1 to 132 for use as a medicament.

134. Use of a pharmaceutical composition according to any one of claims 1 to 132 in the preparation of a medicament for: (i) treating or delaying the progression of a cell proliferative disorder in a subject in need thereof; or (ii) enhancing immune function in a subject suffering from a cell proliferative disorder.

135. A pharmaceutical composition according to any one of claims 1 to 132, for use in: (i) treating or delaying the progression of a cell proliferative disorder in a subject in need thereof; or (ii) enhancing immune function in a subject suffering from a cell proliferative disorder.

136. The use or pharmaceutical composition for use of claim 134 or 135, wherein the cell proliferative disorder is cancer.

137. The pharmaceutical composition for use or use according to any one of claims 133 to 136, wherein the therapeutic protein is a bispecific antibody formulated to bind to a CD3 molecule located on an immune effector cell and a target molecule located on a target cell other than the immune effector cell.

138. The use or pharmaceutical composition for use according to claim 137, wherein the bispecific antibody activates the immune effector cell after binding to the CD3 molecule and to the target molecule.

139. The pharmaceutical composition for use or use according to claim 138, wherein the activated immune effector cells are capable of exerting cytotoxic and / or apoptotic effects on the target cells.

140. The use of a pharmaceutical composition according to any one of claims 1 to 57, 78 to 87 and 90 to 132 in the preparation of a medicament for treating a cell proliferative disorder or delaying its progression, or for enhancing the immune function of a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), B-cell lymphoma, splenic diffuse red pulp small B-cell lymphoma, a type II lymphoma intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma. B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and typical Hodgkin 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, primary DLBCL of the central nervous system, primary cutaneous DLBCL (leg type), Epstein-Barr virus (EBV)-positive elderly DLBCL, 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 caused by HHV8-related multicentric Castleman disease, B-cell leukemia, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system Lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, variant hairy cell leukemia, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granuloma, plasmablastic lymphoma, and primary effusion lymphoma.

141. A pharmaceutical composition according to any one of claims 1 to 57, 78 to 87 and 90 to 132 for use in treating or delaying the progression of a cell proliferative disorder, or for use in enhancing immune function in a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of NHL, 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, DLBCL, GCB DLBCL, 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), 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 caused by HHV8-related multicentric Castleman disease, B cell leukemia, FL, MCL , AML, MZL, SLL, LL, WM, CNSL, BL, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, variant hairy cell leukemia, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, MALT lymphoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granuloma, plasmablastic lymphoma, and primary effusion lymphoma.

142. The use or pharmaceutical composition for use according to claim 140 or 141, wherein the cancer is GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL or BL.

143. Use of a pharmaceutical composition according to any one of claims 1 to 57, 68 to 87 and 89 to 132 in the preparation of a medicament for treating a cell proliferative disorder or delaying its progression, or for enhancing immune function in a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, multiple myeloma (MM), renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma and glioblastoma.

144. A pharmaceutical composition according to any one of claims 1 to 57, 68 to 87 and 89 to 132, for use in treating or delaying the progression of a cell proliferative disorder, or for use in enhancing immune function in a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma and glioblastoma.

145. The use or pharmaceutical composition for use according to claim 143 or 144, wherein the cancer is a HER2 positive cancer.

146. Use of a pharmaceutical composition according to any one of claims 1 to 67, 78 to 88 and 90 to 132 in the preparation of a medicament for treating a cell proliferative disorder or delaying its progression, or for enhancing immune function in a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of MM, CLL, MCL, DLBCL, FL, AML, myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia (APL), chronic myeloproliferative disorder, platelet leukemia, precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursor T-cell acute lymphoblastic leukemia (pre-T-ALL), mastocytosis, mast cell leukemia, mast cell sarcoma, myeloid sarcoma, lymphoid leukemia and undifferentiated leukemia.

147. A pharmaceutical composition according to any one of claims 1 to 67, 78 to 88 and 90 to 132 for use in treating or delaying the progression of a cell proliferative disorder, or for use in enhancing the immune function of a subject suffering from a cell proliferative disorder, wherein the cell proliferative disorder is a cancer selected from the group consisting of MM, CLL, MCL, DLBCL, FL, AML, MDS, CML, chronic myelomonocytic leukemia, APL, chronic myeloproliferative disorder, platelet-derived leukemia, pre-B-ALL, pre-T- ALL, mast cell disease, mast cell leukemia, mast cell sarcoma, myeloid sarcoma, lymphoid leukemia, and undifferentiated leukemia.

148. The use or pharmaceutical composition for use according to claim 146 or 147, wherein the cancer is a FcRH5 positive cancer.

149. The use or pharmaceutical composition for use of any one of claims 134 to 148, wherein the therapeutic protein is a bispecific antibody formulated for administration to the subject at a dose of about 10 μg to about 100 mg.

150. The pharmaceutical composition for use or use of claim 149, wherein the therapeutic protein is a bispecific antibody formulated for administration to the subject at a dose of about 1 mg to about 60 mg.

151. The pharmaceutical composition for use or use of any one of claims 134 to 150, wherein at least one additional therapeutic agent is to be co-administered to the subject.

152. The use or pharmaceutical composition for use according to any one of claims 134 to 151, wherein the subject is a human.

153. A method of treating or delaying progression of a cell proliferative disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 132.

154. A method of enhancing immune function in a subject suffering from a cell proliferative disorder, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 132.

155. The method of claim 153 or 154, wherein the cell proliferative disorder is cancer.

156. The method of any one of claims 153 to 155, wherein the therapeutic protein is a bispecific antibody formulated to bind to a CD3 molecule located on an immune effector cell and a target molecule located on a target cell other than the immune effector cell.

157. The method of claim 156, wherein the bispecific antibody activates the immune effector cell upon binding to the CD3 molecule and to the target molecule.

158. The method of claim 157, wherein the activated immune effector cells are capable of exerting cytotoxic and / or apoptotic effects on the target cells.

159. A method of treating a cell proliferative disorder in a subject in need thereof or delaying its progression or enhancing immune function in a subject with a cell proliferative disorder, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 57, 78 to 87 and 90 to 132, wherein the cell proliferative disorder is a cancer selected from the group consisting of NHL, 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, DLBCL, GCB DLBCL, 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), 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 caused by HHV8-related multicentric Castleman disease, B cell leukemia, FL, MCL , AML, MZL, SLL, LL, WM, CNSL, BL, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, variant hairy cell leukemia, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, MALT lymphoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, lymphomatoid granuloma, plasmablastic lymphoma, and primary effusion lymphoma.

160. The method of claim 159, wherein the cancer is GCB DLBCL, ABC DLBCL, FL, MCL, AML, CLL, MZL, SLL, LL, WM, CNSL, or BL.

161. A method of treating a cell proliferative disorder in a subject in need thereof or delaying its progression or enhancing immune function in a subject with a cell proliferative disorder, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 57, 68 to 87, and 89 to 132, wherein the cell proliferative disorder is a cancer selected from the group consisting of breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and glioblastoma.

162. The method of claim 161, wherein the cancer is a HER2-positive cancer.

163. A method of treating a cell proliferative disorder in a subject in need thereof or delaying its progression or enhancing immune function in a subject with a cell proliferative disorder, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 67, 78 to 88 and 90 to 132, wherein the cell proliferative disorder is a cancer selected from the group consisting of MM, CLL, MCL, DLBCL, FL, AML, MDS, CML, chronic myelomonocytic leukemia, APL, chronic myeloproliferative disorder, platelet-derived leukemia, pre-B-ALL, pre-T-ALL, mastocytosis, mast cell leukemia, mast cell sarcoma, myeloid sarcoma, lymphoid leukemia, and undifferentiated leukemia.

164. The method of claim 163, wherein the cancer is a FcRH5-positive cancer.

165. The method of any one of claims 153 to 164, wherein the therapeutic protein is a bispecific antibody and is administered to the subject at a dose of about 10 μg to about 100 mg.

166. The method of claim 165, wherein the therapeutic protein is administered to the subject at a dose of about 1 mg to about 60 mg.

167. The method of any one of claims 153 to 166, wherein at least one additional therapeutic agent is co-administered to the subject.

168. The method of any one of claims 153 to 167, wherein the subject is a human.

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