Methods for reducing aggregation of bispecific antibodies

Abstract

The invention disclosed herein relates to methods for reducing aggregation of bispecific antibodies, for example, reducing aggregation of bispecific T cell engager (BiTE) antibody constructs that occurs as a result of storage under frozen conditions, by maintaining the antibodies at a certain temperature after thawing.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 739,542, filed October 1, 2018, which is incorporated herein by reference.

[0002] The present application relates to methods for reducing aggregation of bispecific antibodies, more specifically, the present application relates to methods for reducing aggregation of bispecific antibodies disclosed herein, such as bispecific T cell engager antibodies, that occurs as a result of storage under frozen conditions. [Background technology]

[0003] Therapeutic proteins, such as antibodies, are an important class of pharmaceutical drugs that serve patients. Typically, therapeutic proteins are produced and purified in eukaryotic cells in large quantities. Because proteins are sensitive to temperature changes, drug substances of these therapeutic proteins are stored and transported under frozen conditions at temperatures ranging from -20°C to -80°C. Freezing extends the shelf life of the therapeutic proteins and allows flexibility in setting up the final formulation and filling the proteins into commercial product packaging. Logistics and shipping for operational infrastructure and storage are preferred at the higher temperatures of the range.

[0004] Freezing / thawing processes can be a source of stress to proteins. For example, during the freezing process of a protein drug substance, water can crystallize, which can cause protein molecules to reach concentration levels several times higher than the initial level. As a result of the concentration change, the thermodynamic stability of the protein can be compromised, which can lead to unfolding events and cause aggregation. See, for example, 18 Lam Philippe et al., Quality by Design for Biopharmaceutical Drug Product Development, pp. 159-189 (Jameel F. et al. ed., 2015). Similar stresses can also be involved in the thawing process, which can be severe since higher temperatures are required to melt the ice in a timely manner. Ibid. Thus, protein aggregates (e.g., high molecular weight (HMW) aggregates) can result from the freeze / thaw process when a drug substance containing a therapeutic protein is stored and / or shipped under frozen conditions. However, the presence of aggregates in a drug substance is undesirable, as aggregates can adversely affect protein stability, immunogenicity and efficacy. What is needed is a method to reduce protein aggregation that results from storage under frozen conditions. Summary of the Invention [Means for solving the problem]

[0005] Provided herein are methods for reducing aggregation of bispecific antibodies, particularly bispecific T cell engager antibodies (BiTEs), as a result of storage under frozen conditions. This application is directed to the surprising finding that maintaining a bispecific antibody at a certain temperature for a period of time after thawing reduces aggregates formed during storage under frozen conditions.

[0006] In one embodiment, disclosed herein is a method for reducing bispecific antibody aggregates comprising maintaining the thawed bispecific antibody at a temperature of about 5° C. to about 45° C. for at least 4 hours, where the bispecific antibody was stored at a temperature of about −20° C. to about −40° C. prior to thawing. In one embodiment, the thawed bispecific antibody is maintained at that temperature for about 4 hours to about 96 hours. In one embodiment, the thawed bispecific antibody is maintained at a temperature of about 10° C. to about 30° C. for about 10 hours to about 48 hours. In one embodiment, the thawed bispecific antibody is maintained at a temperature of about 10° C. to about 30° C. for 8 hours to 48 hours.

[0007] In one embodiment, the bispecific antibody is thawed at a temperature of about 5° C. to about 45° C. In one embodiment, the bispecific antibody was stored at a temperature of about −20° C. to about −35° C. prior to thawing, and in another embodiment, the bispecific antibody was stored at about −30° C. prior to thawing. In one embodiment, the aggregates comprise high molecular weight (HMW) aggregates. In one embodiment, the HMW aggregates comprise dimers of the bispecific antibody. In one embodiment, the bispecific antibody comprises less than about 1% HMW aggregates after the storage period, and in another embodiment, the bispecific antibody comprises less than about 0.5% HMW aggregates after the storage period.

[0008] In one embodiment, disclosed herein is a method for preparing a composition comprising a bispecific antibody, comprising thawing a drug substance comprising a bispecific antibody stored at a temperature of about -20°C to about -40°C and holding the thawed drug substance comprising a bispecific antibody at a temperature of about 5°C to about 45°C for at least 4 hours. In one embodiment, the drug substance is held at that temperature for about 4 hours to about 96 hours. In another embodiment, the drug substance is held at a temperature of about 10°C to about 30°C for about 10 hours to about 48 hours. In another embodiment, the drug substance is held at a temperature of about 10°C to about 30°C for 8 hours to 48 hours. In one embodiment, the drug substance is thawed at a temperature of about 5°C to about 45°C. In one embodiment, the drug substance is stored at a temperature of about -20°C to about -35°C, and in another embodiment, the drug substance was stored at about -30°C. In one embodiment, the method for preparing the composition further comprises filtering the drug substance, and in another embodiment, the method for preparing the composition further comprises dividing the composition into dosage forms. In one embodiment, the composition is a pharmaceutical composition comprising a bispecific antibody.

[0009] In one embodiment, a method for preparing a composition comprising a bispecific antibody is disclosed herein, the method comprising holding a thawed drug substance comprising a bispecific antibody at a temperature of about 5° C. to about 45° C. for at least 4 hours, the drug substance having been frozen at or above the glass transition temperature (Tg') of the drug substance prior to thawing. In one embodiment, the drug substance is frozen at a temperature of about -10° C. to or above the glass transition temperature of the drug substance, and in another embodiment, the drug substance was frozen at about -32° C. In one embodiment, the drug substance is held at that temperature for about 4 hours to about 96 hours. In one embodiment, the drug substance is thawed at a temperature of about 5° C. to about 45° C. In another embodiment, the drug substance is held at the same temperature at which the drug substance is thawed. In another embodiment, the drug substance is thawed and held at the same temperature of about 15° C. to about 30° C. for 30 hours to 50 hours. In another embodiment, the method for preparing the composition further comprises dividing the composition into formulations. In another embodiment, the method for preparing the composition further comprises lyophilizing the composition. In yet another embodiment, the method for preparing the composition further comprises spray drying the composition.

[0010] In one embodiment, the drug substance contains less than about 1% HMW aggregates after the holding period, and in another embodiment, the drug substance contains less than about 0.5% HMW aggregates, hi one embodiment, the HMW aggregates comprise dimers of the bispecific antibody.

[0011] In one embodiment, the drug substance comprises the bispecific antibody at a concentration of about 0.05 mg / mL to about 20 mg / mL.

[0012] In one embodiment, the bispecific antibody is a bispecific T cell engager antibody (BiTE). In one embodiment, the bispecific antibody comprises a first binding domain that binds to a target cell surface antigen and a second binding domain that binds to human CD3, and the bispecific antibody is in an (scFv)2 format. In one embodiment, the bispecific antibody comprises a first binding domain that binds to a target cell surface antigen selected from CD19, CD33 or BCMA, and a second binding domain that binds to human CD3, and the bispecific antibody is in an (scFv)2 format.

[0013] In one embodiment, the bispecific antibody comprises a first binding domain and a second binding domain, the first binding domain comprising a VH region and a VL region, wherein the VH comprises the amino acid sequence of SEQ ID NO: 77 and the VL comprises the amino acid sequence of SEQ ID NO: 78; or the VH comprises the amino acid sequence of SEQ ID NO: 28 and the VL comprises the amino acid sequence of SEQ ID NO: 32 or 33; or the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO: 133. In another embodiment, the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 17, 40 or 135.

[0014] In one embodiment, the bispecific antibody is a BiTE, the BiTE further comprising a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, the two polypeptide monomers being linked together via a peptide linker. In another embodiment, the third domain comprises, in amino to carboxyl order, hinge-CH2-CH3-linker-hinge-CH2-CH3. In one embodiment, the third domain is a half-life extension (HLE) domain.

[0015] In one embodiment, the bispecific antibody comprises a first binding domain, a second binding domain and a third domain, wherein the first binding domain binds to at least one target cell surface antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA or PSMA, the second binding domain binds to human CD3, and the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, and the two polypeptide monomers are linked to each other via a peptide linker.

[0016] In one embodiment, the bispecific antibody comprises a first binding domain, a second binding domain and a third domain, wherein the first binding domain comprises a VH region and a VL region and binds to at least one target cell surface antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA or PSMA, the second binding domain binds to human CD3, and the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, the two polypeptide monomers being linked to each other via a peptide linker, wherein: (a) the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 109; or (b) the VH comprises the amino acid sequence of SEQ ID NO: 27 and the VL comprises the amino acid sequence of SEQ ID NO: 32; or (c) the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 49; or (d) the VH comprises the sequence or (e) the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO:78; or (f) the VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO:112; or (g) the VH comprises the amino acid sequence of SEQ ID NO:89 and the VL comprises the amino acid sequence of SEQ ID NO:90; or (h) the VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO: or (i) the VH comprises the amino acid sequence of SEQ ID NO: 121 and the VL comprises the amino acid sequence of SEQ ID NO: 122; or (j) the VH comprises the amino acid sequence of SEQ ID NO: 188 and the VL comprises the amino acid sequence of SEQ ID NO: 189; or (k) the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO: 133; or (l) the VH comprises the amino acid sequence of SEQ ID NO: 173 and the VL comprises the amino acid sequence of SEQ ID NO: 174.

[0017] In one embodiment, the bispecific antibody comprises a first binding domain, a second binding domain and a third domain, wherein the first binding domain binds to at least one target cell surface antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA or PSMA, the second binding domain binds to human CD3, and the third domain comprises two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker, and the bispecific antibody comprises or consists of an amino acid sequence selected from SEQ ID NOs: 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

[0018] In one embodiment, the bispecific antibody is a masked bispecific antigen-binding protein. In one embodiment, a masked bispecific antigen-binding protein comprises (a) a first antibody or antigen-binding fragment thereof (AB1) that binds to a first antigen and a masking domain (MD1) coupled to AB1, where MD1 comprises (1) a first masking peptide (MP1) that inhibits or reduces binding of AB1 to its antigen and (2) a protein recognition site (PR1), such that binding to or cleavage of PR1 by a protein or protease increases AB1 binding to its antigen. (b) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen and a second masking domain (MD2) coupled to AB2, wherein MD2 comprises (1) a second masking peptide (MP) that inhibits or reduces binding of AB2 to the antigen and (2) a second protein recognition site (PR2), such that binding to or cleavage of PR2 by a protein or protease increases AB2 binding to the antigen. In one embodiment, PR1 and PR2 comprise the same protein recognition sequence. In another embodiment, AB1 binds human CD3 and AB2 binds human EGFR. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the increase in aggregate levels (ΔHMW%) after storage of various HLE BiTE molecules at −20° C. for 1 month. [Figure 2A] FIG. 2A shows the increase in aggregate levels (ΔHMW%) of DLL3xCD3 HLE BiTE after one month of storage at −20° C. in compositions with different pH. [Figure 2B] FIG. 2B shows the increase in aggregate levels (ΔHMW%) of DLL3xCD3 HLE BiTE after storage at different temperatures for 1 month. [Figure 3A] Figures 3A, 3B, 3C, and 3D show that HMW aggregate levels increased in various HLE BiTEs after storage at -20°C or -30°C, and that holding at room temperature for 24 hours after thawing reduced aggregate levels. [Figure 3B] Figures 3A, 3B, 3C, and 3D show that HMW aggregate levels increased in various HLE BiTEs after storage at -20°C or -30°C, and that holding at room temperature for 24 hours after thawing reduced aggregate levels. [Figure 3C] Figures 3A, 3B, 3C, and 3D show that HMW aggregate levels increased in various HLE BiTEs after storage at -20°C or -30°C, and that holding at room temperature for 24 hours after thawing reduced aggregate levels. [Figure 3D] Figures 3A, 3B, 3C, and 3D show that HMW aggregate levels increased in various HLE BiTEs after storage at -20°C or -30°C, and that holding at room temperature for 24 hours after thawing reduced aggregate levels. [Figure 4] FIG. 4 shows the dependence of the decrease in HMW level of BiTE molecules on the holding time and holding temperature. [Diagram 5] FIG. 5 shows the stabilizing effect of benzyl alcohol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] method Described herein are methods for reducing aggregates of bispecific antibodies, particularly aggregates formed when bispecific antibodies are stored under frozen conditions. As used herein, the term "aggregates" or "aggregation" refers to the association of two or more molecules. In certain embodiments, aggregates are high molecular weight (HMW) aggregates having a larger molecular weight and / or size than non-aggregated molecules. In certain embodiments, aggregates comprise the association of two or more antibody molecules. In certain embodiments, aggregates comprise the association of two or more bispecific antibody molecules, including bispecific antibody dimers. In certain embodiments, the bispecific antibody is a bispecific T cell engager antibody (BiTE).

[0021] The presence and / or level of aggregates may be determined by techniques known in the art, such as techniques that determine the size of molecules, such as size exclusion chromatography, cation exchange chromatography, X-ray diffraction, modulated differential scanning calorimetry (mDSC) and non-denaturing polyacrylamide gel electrophoresis (PAGE). In one embodiment, the presence and / or level of protein aggregates is determined by size exclusion HPLC (SE-HPLC). In another embodiment, the presence and / or level of protein aggregates is determined by size exclusion high performance HPLC (SE-UHPLC).

[0022] In one aspect, a method for reducing bispecific antibody aggregates is disclosed herein, comprising maintaining a thawed bispecific antibody at a temperature for at least 4 hours, the bispecific antibody having been stored under frozen conditions prior to thawing. As used herein, the term "thawed bispecific antibody" or "thawed drug substance comprising a bispecific antibody" is understood to refer to a bispecific antibody in a liquid state or a drug substance comprising a bispecific antibody in a liquid state resulting from exposure to heat. In certain embodiments, a thawed bispecific antibody or a thawed drug substance comprising a bispecific antibody is a bispecific antibody or a drug substance comprising a bispecific antibody in a liquid state that is free or substantially free of frozen material.

[0023] In certain embodiments, the bispecific antibody is a drug substance. As used herein, the term "drug substance" is understood to refer to a recombinant protein (e.g., a bispecific antibody) that has been sufficiently purified or isolated from contaminating proteins, lipids and nucleic acids (e.g., contaminating proteins, lipids and nucleic acids present in a liquid medium or derived from a host cell (e.g., derived from a mammalian, yeast or bacterial host cell)) and biological contaminants (e.g., viral and bacterial contaminants) and can be formulated into a pharmaceutical composition without further substantial purification and / or contaminant removal steps. The term drug substance encompasses compositions that include a sufficiently purified recombinant protein (e.g., a bispecific antibody) and one or more pharma- ceutically suitable excipients.

[0024] In certain embodiments, the thawed drug substance is a composition comprising one or more excipients in addition to the recombinant protein (e.g., bispecific antibody). Appropriate excipients may be used as appropriate for the pharmaceutical composition. Exemplary excipients include a buffer (e.g., acetate buffer, glutamate buffer, citrate buffer, lactate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer or phosphate buffer), a sugar (e.g., glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol or xylitol) and a surfactant (e.g., polysorbate 20 or polysorbate 80). In certain embodiments, the thawed drug substance is a composition comprising the recombinant protein (e.g., bispecific antibody), a buffer (e.g., glutamate buffer or citrate buffer), a sugar (e.g., sucrose) and optionally a surfactant (e.g., polysorbate 80). The pH of the thawed drug substance may range from about 3.0 to 7.0, or from about 4.0 to about 6.0.

[0025] In certain embodiments, the frozen bispecific antibody is thawed by exposure to elevated temperatures. In certain embodiments, the bispecific antibody is thawed at a temperature of about 0° C. to about 50° C., or about 0° C. to about 40° C., or about 0° C. to about 30° C., or about 5° C. to about 45° C., or about 5° C. to about 30° C., or about 10° C. to about 50° C., or about 10° C. to about 40° C., or about 10° C. to about 30° C., or about 15° C. to about 50° C., or about 15° C. to about 40° C., or about 15° C. to about 30° C., or about 20° C. to about 30° C., or about 25° C. to about 30° C. In certain embodiments, the bispecific antibody is thawed at a temperature of about 0° C. to about 25° C., or about 5° C. to about 25° C., or about 10° C. to about 25° C., or about 15° C. to about 25° C., or about 20° C. to about 25° C. In certain embodiments, the bispecific antibody is thawed at a temperature of about 0° C., about 5° C., about 10° C., about 15° C., about 20° C., about 25° C., or about 30° C., or about 40° C., or about 45° C., or about 50° C. As can be appreciated by one of skill in the art, the bispecific antibody may be gently mixed during thawing to ensure uniform temperature distribution and / or disruption of concentration gradients formed during thawing. Gentle mixing may be achieved, for example, by using a tilting shaker or by gently tumbling a container of the bispecific antibody. Alternatively, the bispecific antibody may be gently mixed after thawing.

[0026] In certain embodiments, the method comprises maintaining the thawed bispecific antibody at a temperature for at least 4 hours. In certain embodiments, the maintaining temperature is from about 0° C. to about 50° C., or from about 0° C. to about 40° C., or from about 5° C. to about 50° C., or from about 5° C. to about 45° C., or from about 5° C. to about 40° C., or from about 5° C. to about 30° C., or from about 10° C. to about 50° C., or from about 10° C. to about 45° C., or from about 10° C. to about 40° C., or from about 10° C. to about 30° C., or from about 15° C. to about 40° C., or from about 15° C. to about 30° C. In certain embodiments, the maintaining temperature is from about 15° C. to about 25° C. In certain embodiments, the holding temperature is about 5°C, or about 10°C, or about 15°C, or about 17°C, or about 19°C, or about 20°C, or about 23°C, or about 25°C, or about 27°C, or about 30°C, or about 35°C, or about 40°C, or about 45°C. In certain embodiments, the thawed bispecific antibody is maintained at any one of the above temperatures for about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 10 hours to about 120 hours, or about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours, or about 24 hours to about 120 hours, or about 24 hours to about 100 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours.

[0027] In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for about 4 hours to about 120 hours, or about 4 hours to about 100 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 10° C. to about 30° C. for about 4 hours to about 24 hours, or about 10 hours to about 120 hours, or about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 30° C. to about 45° C. for about 4 hours to about 24 hours, or about 4 hours to about 10 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15° C. to about 45° C. for about 4 hours to about 120 hours, or about 4 hours to about 50 hours, or about 4 hours to about 24 hours, or about 8 hours to about 50 hours, or about 15 hours to about 50 hours, or about 4 hours to about 100 hours, or about 8 hours to about 100 hours, or about 15 hours to about 100 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15° C. to about 30° C. for about 4 hours to about 24 hours, or about 10 hours to about 120 hours, or about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours, about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours.In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15° C. to about 25° C. for about 4 hours to about 24 hours, or about 10 hours to about 48 hours, or about 15 hours to about 30 hours, or about 15 hours to about 24 hours, or about 24 hours to about 120 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours. In another embodiment, the thawed bispecific antibody is maintained at a temperature of about 15° C. to about 30° C. for about 10 hours, or about 15 hours, or about 20 hours, or about 24 hours, or about 36 hours, or about 48 hours, or about 60 hours, or about 72 hours, or about 84 hours, or about 96 hours, or about 120 hours.

[0028] The bispecific antibodies were stored under frozen conditions prior to thawing. In certain embodiments, the bispecific antibodies were stored at a temperature of about -20°C to about -50°C. In certain embodiments, the bispecific antibodies were stored at a temperature of about -20°C to about -40°C. In certain embodiments, the bispecific antibodies were stored at a temperature of about -25°C to about -35°C. In certain embodiments, the bispecific antibodies were stored at a temperature of about -20°C, about -30°C, about -35°C, about -40°C, or about -50°C. In certain embodiments, the bispecific antibodies were stored at a temperature of about -30°C. In certain embodiments, the bispecific antibodies were stored at a temperature of 0°C to a temperature equal to or greater than the glass transition temperature (Tg') of the antibody or the Tg' of a composition comprising the antibody. In certain embodiments, the bispecific antibodies were stored at a temperature of about -10°C to a temperature equal to or greater than the Tg' of the antibody or the Tg' of a composition comprising the antibody.

[0029] In certain embodiments, the bispecific antibodies have been stored at a temperature of about -20°C to about -40°C for about 1 day to about 5 years. In certain embodiments, the bispecific antibodies have been stored at a temperature of about -20°C to about -40°C for about 1 week to about 5 years or about 1 month to about 5 years. In certain embodiments, the bispecific antibodies have been stored at a temperature of about -20°C to about -40°C for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 6 months, about 18 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years.

[0030] As used herein, the term "about," when used to modify a particular value or range, is understood to mean that there may be a variation in the given value or range, including up to 20 percent, e.g., 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent above and below the stated value or range.

[0031] As used herein, the term "storing" or "storage" refers to placing or setting aside a bispecific antibody under certain conditions for later use or further processing. In certain embodiments, the antibody is placed under any of the temperatures described above, which may be accomplished, for example, by using a freezer, refrigerated truck, or shipping equipment capable of maintaining the required temperature. As can be appreciated by one of skill in the art, the antibody is frozen when stored under the storage temperatures described above.

[0032] The bispecific antibody may be stored in any suitable container that can maintain its integrity under storage temperature. Exemplary containers include vials, bottles, bags, and carboys. Such containers are well known in the art and commercially available. In certain embodiments, the bispecific antibody is stored in a disposable container, such as a commercially available flexible freeze thaw container, such as the Celsius® FFT system. The volume of the bispecific antibody under storage is determined by the volume of the container used for storage. In certain embodiments, the volume of the container is 5 mL, 10 mL, 100 mL, 500 mL, 1 liter (L), 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, or 12 L. In certain embodiments, the container of bispecific antibody under storage is about 5 mL, about 10 mL, about 100 mL, about 500 mL, about 1 liter (L), about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, or about 12 L.

[0033] In certain embodiments, the concentration of the bispecific antibody during storage ranges from about 0.01 mg / mL to about 25 mg / mL, or from about 0.05 mg / mL to about 25 mg / mL, or from about 0.1 mg / mL to about 25 mg / mL, or from about 0.5 mg / mL to about 25 mg / mL, or from about 1 mg / mL to about 25 mg / mL. In certain embodiments, the concentration of the bispecific antibody during storage ranges from about 1 mg / mL to about 20 mg / mL, or from about 1 mg / mL to about 15 mg / mL, or from about 1 mg / mL to about 10 mg / mL, or from about 1 mg / mL to about 5 mg / mL. In certain embodiments, the concentration of the bispecific antibody under storage is about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, or about 25 mg / mL.

[0034] Also disclosed herein are methods for preparing a composition comprising a bispecific antibody. In certain embodiments, the methods comprise thawing a drug substance comprising a bispecific antibody that has been stored in a frozen state and maintaining the thawed drug substance at a temperature for at least 4 hours.

[0035] In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C to about -50°C. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C to about -35°C. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -25°C to about -35°C. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C, about -30°C, about -35°C, about -40°C, or about -50°C. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -30°C.

[0036] In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C to about -50°C for about 1 day to about 5 years. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C to about -40°C for about 1 week to about 5 years or about 1 month to about 5 years. In certain embodiments, the bispecific antibody-containing drug substance has been stored at a temperature of about -20°C to about -40°C for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 6 months, about 18 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years.

[0037] In certain embodiments, a method for preparing a composition comprising a bispecific antibody comprises holding a thawed drug substance comprising a bispecific antibody at a temperature for at least 4 hours, the drug substance having been frozen at a temperature equal to or greater than the glass transition temperature (Tg') of the drug substance prior to thawing. In certain embodiments, the drug substance comprising a bispecific antibody has been frozen at a temperature equal to or greater than about -10°C to a temperature equal to or greater than the Tg' of the drug substance. In certain embodiments, the drug substance comprising a bispecific antibody has been frozen at a temperature equal to or greater than about -20°C to a temperature equal to or greater than the Tg' of the drug substance. In certain embodiments, the drug substance comprising a bispecific antibody has been frozen at a temperature equal to or greater than the Tg' of the drug substance (e.g., from about -10°C to Tg') for about 1 day to about 5 years or about 1 week to about 5 years. In certain embodiments, the drug substance comprising a bispecific antibody has been frozen at a temperature equal to or greater than the Tg' of the drug substance (e.g., from about -10°C to Tg') for about 1 month, about 6 months, about 1 year, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years. In certain embodiments, the Tg' of the drug substance is about -30° C., or about -32° C., or about -35° C. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature that is about -32° C. for about 1 month, about 6 months, about 1 year, about 18 months, about 2 years, about 3 years, about 4 years, or about 5 years.

[0038] As can be appreciated by one of skill in the art, the glass transition temperature of a bispecific antibody or a drug substance comprising a bispecific antibody can be determined by methods known in the art, such as, for example, differential scanning calorimetry (DSC), thermomechanical analysis (TMA), dynamic mechanical analysis (DMA), and dilatometry.

[0039] The bispecific antibody-containing drug substance may be stored or frozen in any suitable container that can maintain its integrity under storage / freezing temperatures. Exemplary containers include vials, bottles, bags, and carboys. Such containers are well known in the art and commercially available. In certain embodiments, the bispecific antibody-containing drug substance is stored in a disposable container, such as a commercially available flexible freeze thaw container, such as the Celsius® FFT system. The volume of the bispecific antibody-containing drug substance under storage is determined by the volume of the container used for storage. In certain embodiments, the volume of the container is 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, or 12L.

[0040] The drug substance, including the bispecific antibody, may be thawed by exposure to elevated temperatures. In certain embodiments, the drug substance is thawed at a temperature of about 0° C. to about 50° C., or about 0° C. to about 40° C., or about 0° C. to about 30° C., or about 5° C. to about 45° C., or about 5° C. to about 30° C., or about 10° C. to about 30° C., or about 15° C. to about 30° C., or about 20° C. to about 30° C., or about 25° C. to about 30° C. In certain embodiments, the drug substance is thawed at a temperature of about 0° C. to about 25° C., or about 5° C. to about 25° C., or about 10° C. to about 25° C., or about 15° C. to about 25° C., or about 20° C. to about 25° C. In certain embodiments, the drug substance is thawed at a temperature of about 0°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, about 27°C, or about 30°C, or about 40°C, or about 45°C, or about 50°C.

[0041] In certain embodiments, the method comprises maintaining the thawed drug substance at a temperature of about 0° C. to about 50° C., or about 0° C. to about 40° C., or about 5° C. to about 50° C., or about 5° C. to about 45° C., or about 10° C. to about 50° C., or about 10° C. to about 45° C., or about 10° C. to about 30° C., or about 15° C. to about 40° C., or about 15° C. to about 30° C. In certain embodiments, the thawed drug substance is maintained at a temperature of about 15° C. to about 25° C. In certain embodiments, the thawed drug substance is maintained at a temperature of about 5° C., or about 10° C., or about 15° C., or about 17° C., or about 19° C., or about 21° C., or about 23° C., or about 25° C., or about 27° C., or about 29° C., or about 40° C., or about 45° C. In certain embodiments, the thawed drug substance is thawed at any one of the above temperatures for about 4 hours to about 150 hours, or about 4 hours to about 100 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 10 hours to about 150 hours, or about 10 hours to about 100 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours. or for about 24 hours to about 150 hours, or for about 24 hours to about 120 hours, or for about 24 hours to about 100 hours, or for about 24 hours to about 96 hours, or for about 24 hours to about 72 hours, or for about 24 hours to about 48 hours, for about 48 hours to about 100 hours, or for about 48 hours to about 96 hours, or for about 48 hours to about 72 hours, or for about 72 hours to about 100 hours, or for about 72 hours to about 96 hours.

[0042] In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for at least about 4 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10° C. to about 45° C. for about 4 hours to about 100 hours, or about 4 hours to about 50 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15° C. to about 150 hours, or about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 10 hours to about 120 hours, or about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15° C. to about 30° C. for about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15° C. to about 25° C. for about 15 hours to about 96 hours, or about 15 hours to about 72 hours, or about 15 hours to about 48 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 100 hours, or about 48 hours to about 96 hours, or about 48 hours to about 72 hours, or about 72 hours to about 100 hours, or about 72 hours to about 96 hours.In one embodiment, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10° C. to about 30° C. for about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours, or about 24 hours to about 96 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours, or about 48 hours to about 96 hours, or about 72 hours to about 96 hours. In another embodiment, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10° C. to about 30° C. for about 4 hours, or about 10 hours, or about 24 hours, or about 48 hours, or about 60 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 150 hours.

[0043] In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for a period of time such that the level of aggregates in the drug substance comprising the bispecific antibody is reduced to approximately the same level as before storage under frozen conditions. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for a period of time ranging from about 4 hours to a time at which the level of aggregates in the drug substance comprising the bispecific antibody is reduced to approximately the same level as before storage under frozen conditions. As can be appreciated by one of skill in the art, the time required to reduce the level of aggregates in the thawed drug substance to approximately the same level as before storage under frozen conditions at a particular temperature is known in the art and can be determined by measuring the level of aggregates in the drug substance before storage under frozen conditions and at various time points after thawing using methods known in the art and used (e.g., SE-UHPLC), see, e.g., Example 5.

[0044] In certain embodiments, the drug substance is held at the same temperature at which the drug substance is thawed. For example, a drug substance comprising a bispecific antibody may be thawed at any of the thawing and holding temperatures disclosed above, such as, for example, at a temperature of about 5° C. to about 45° C., and then held at the same temperature for at least 4 hours after thawing (e.g., about 4 hours to about 150 hours, or about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 8 hours to about 96 hours, or about 8 hours to about 72 hours, or about 8 hours to about 48 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours). Whether a drug substance is thawed can be readily determined by one of skill in the art.

[0045] In certain embodiments, the drug substance is held at the same temperature at which the drug substance is thawed, and the drug substance is held at the same temperature for the entire time (time for thawing and holding) until the level of aggregates in the drug substance, including the bispecific antibody, has decreased to approximately the same level as before storage under frozen conditions. As shown in the Examples, one of skill in the art may determine the level of aggregates in the drug substance at various times before freezing and after thawing using methods known in the art, such as SE-UHPLC. In one embodiment, the drug substance is thawed and held at the same temperature, from about 5° C. to about 45° C., for a total time of about 30 hours to about 100 hours, or for a total time of about 30 hours to about 90 hours, or for a total time of about 30 hours to about 80 hours, or for a total time of about 30 hours to about 70 hours, or for a total time of about 30 hours to about 60 hours, or for a total time of about 30 hours to about 50 hours. In one embodiment, the drug substance is thawed and maintained at the same temperature, between about 15° C. and about 30° C., for a total time of about 30 hours to about 100 hours, or for a total time of about 30 hours to about 90 hours, or for a total time of about 30 hours to about 80 hours, or for a total time of about 30 hours to about 70 hours, or for a total time of about 30 hours to about 60 hours, or for a total time of about 30 hours to about 50 hours.

[0046] In certain embodiments, the drug substance comprising the bispecific antibody is gently mixed during thawing. Gentle mixing can be achieved, for example, by using a tilting shaker or by gently tumbling the drug substance container. In certain embodiments, the drug substance comprising the bispecific antibody is not mixed during thawing, instead the drug substance is gently mixed after thawing.

[0047] In certain embodiments, the drug substance comprises the bispecific antibody at a concentration of about 0.01 mg / mL to about 25 mg / mL, or about 0.05 mg / mL to about 25 mg / mL, about 0.1 mg / mL to about 25 mg / mL, about 0.5 mg / mL to about 25 mg / mL, or about 1 mg / mL to about 25 mg / mL. In certain embodiments, the drug substance comprises the bispecific antibody at a concentration of about 1 mg / mL to about 20 mg / mL, or about 1 mg / mL to about 15 mg / mL, or about 1 mg / mL to about 10 mg / mL, or about 1 mg / mL to about 5 mg / mL. In certain embodiments, the drug substance comprises a bispecific antibody at a concentration of about 0.01 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, or about 25 mg / mL.

[0048] In certain embodiments, the bispecific antibody-containing drug substance has a pH in the range of about pH 3.5 to about pH 7.5 or about pH 4.0 to about pH 7.0. In certain embodiments, the bispecific antibody-containing drug substance has a pH in the range of about pH 4.0 to about pH 6.5. In certain embodiments, the bispecific antibody-containing drug substance has a pH in the range of about pH 4.0 to about pH 4.8. In certain embodiments, the bispecific antibody-containing drug substance has a pH of about 3.5, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about 6.6, about 7.0 or about 7.5.

[0049] In certain embodiments, the method further comprises filtering the drug substance. In certain embodiments, the filtering step comprises sterile filtration. Sterile filtration is well known and commonly used in the art. For example, sterile filtration can be performed using Normal Flow Filtration (NFF), where the direction of fluid flow is perpendicular to the filtration medium (e.g., membrane) and the purified liquid passes through the filtration medium. In certain embodiments, agents that can reduce aggregation of bispecific antibodies, such as amino acids, polyols, such as benzyl alcohol, cyclodextran, dextran, and polyethylene glycol (PEG), can be added to the drug substance.

[0050] In certain embodiments, the composition prepared by the method is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" is understood to refer to a formulation comprising a bispecific antibody suitable for injection and / or administration to a patient (e.g., a human) in need thereof. More particularly, the pharmaceutical composition is substantially sterile and does not contain any material that is overly toxic or infectious to the recipient.

[0051] In certain embodiments, the method for preparing a composition comprising a bispecific antibody further comprises dividing the composition into formulations. Such formulations may be presented in unit dosage forms, for example in ampoules, single-dose containers, or in multi-dose containers. The formulations may be presented in a vial, pack, or dispenser device, which may contain one or more unit dosage forms containing the bispecific antibody, as appropriate.

[0052] In certain embodiments, the method for preparing a composition comprising a bispecific antibody further comprises lyophilizing the composition. In certain embodiments, the lyophilization step is performed after dividing the composition into formulations. Methods for lyophilizing pharmaceutical compositions are well known and commonly used in the art. See, for example, Cryopreservation and Freeze-Drying Protocols (JG Day and GN Stacey ed., Springer 2017). The lyophilization step can be performed before or after the division step.

[0053] In certain embodiments, the method for preparing a composition comprising a bispecific antibody further comprises spray drying the composition. Methods for spray drying pharmaceutical compositions are well known and commonly used in the art. See, for example, Niven, R., Prestrelski, SJ, Treuheit, MJ, Ip, AY and Arakawa, T. Protein Nebulization II. Stabilization of G-CSF to air-jet nebulization and the role of protectants. (1996) Int. J. Pharm. 127:191-20. The spray drying step can be performed before or after the partitioning step.

[0054] The methods disclosed herein reduce aggregates of bispecific antibodies. The methods are based on the surprising finding that thawed antibodies, when kept at a certain temperature, e.g., about 5°C to about 45°C, for at least 4 hours (e.g., for a period of about 4 hours to about 96 hours), reduce aggregates formed during storage under frozen conditions (e.g., about -20°C to about -40°C). Without wishing to be bound by any theory, it is believed that the aggregates revert to a non-aggregated state after the holding period.

[0055] In certain embodiments, the aggregates comprise HMW aggregates. In certain embodiments, the bispecific antibody comprises less than about 5% or less than about 3% or less than about 2% or less than about 1% or less than about 0.5% HMW aggregates after the holding period. In certain embodiments, the HMW aggregates formed under freezing conditions are reduced to the same level or substantially the same level as before freezing. In certain embodiments, the HMW aggregates comprise bispecific antibody dimers. In certain embodiments, the bispecific antibody comprises less than about 1% bispecific antibody dimers after the holding period. In certain embodiments, the bispecific antibody comprises less than about 0.5% bispecific antibody dimers after the holding period.

[0056] In certain embodiments, the drug substance comprising a bispecific antibody contains less than about 5% or less than about 3% or less than about 2% or less than about 1% or less than about 0.5% HMW aggregates after the holding period. In certain embodiments, the HMW aggregates comprise bispecific antibody dimers. In certain embodiments, the drug substance comprising a bispecific antibody contains less than about 1% bispecific antibody dimers after the holding period. In certain embodiments, the drug substance comprising a bispecific antibody contains less than about 0.5% bispecific antibody dimers after the holding period.

[0057] The methods disclosed herein do not affect or do not substantially affect the stability of the bispecific antibody or bispecific antibody-containing drug substance. In certain embodiments, the methods produce a bispecific antibody or bispecific antibody-containing drug substance that has the same or substantially the same color and / or transparency compared to the bispecific antibody or bispecific antibody before freezing. In certain embodiments, the methods produce a bispecific antibody or bispecific antibody-containing drug substance that has the same or substantially the same charge variant compared to the bispecific antibody or bispecific antibody before freezing. In certain embodiments, the methods produce a bispecific antibody or bispecific antibody-containing drug substance that has the same or substantially the same potency compared to the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or bispecific antibody-containing drug substance that has the same or substantially the same clipping level compared to the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or bispecific antibody-containing drug substance that has the same or substantially the same chemical modification (e.g., glycosylation) compared to the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or a bispecific antibody-comprising drug substance that has the same or substantially the same pH compared to the bispecific antibody or bispecific antibody-comprising drug substance prior to freezing.

[0058] bispecific antibody Bispecific antibodies that may be used in the methods disclosed herein include those that tend to aggregate under frozen conditions, e.g., at temperatures ranging from −20° C. to −50° C., e.g., due to hydrophobic interactions between different regions of the antibody. As used herein, the term “bispecific antibody” is understood to refer to an antibody capable of specifically binding to two different antigens or targets or epitopes. In certain embodiments, a bispecific antibody comprises a first domain that specifically binds to one antigen or target and a second domain that specifically binds to another antigen or target. In certain embodiments, the first domain of the bispecific antibody specifically binds to a target cell surface antigen and the second binding domain of the bispecific antibody specifically binds to human CD3, a subunit of the T cell receptor complex on T cells. In certain preferred embodiments, the bispecific antibody is a bispecific T cell engager (BiTE) antibody construct. See, e.g., WO2008119567 and WO2017134140.

[0059] As used herein below, the term "domain specifically binds" or "domain that binds" is understood to refer to a domain that specifically binds to / interacts with / recognizes a certain target or epitope. The binding domain of an antibody construct comprises the minimum structural requirements of an antibody that allows target binding. This minimum requirement may be defined by the presence of at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the light chain variable region (VL) region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) region), preferably all six CDRs. Preferably, these CDRs are comprised in the antibody VL and antibody VH frameworks. The term includes fragments of full-length antibodies and antibody variants. Examples of antibody fragments, antibody variants or binding domains include: (1) Fab fragment, a monovalent fragment having VL, VH, CL and CH1 domains; (2) F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) Fd fragment having two VH and CH1 domains; (4) Fv fragment having the VL and VH domains of a single arm of an antibody, (5) dAb fragment (Ward et al., (1989) Nature 341:544-546), having a VH domain; (6) isolated complementarity determining regions (CDRs) and (7) single chain Fv (scFv), the latter being preferred (e.g. from an scFv-library). Further antibody fragments include VH, VHH, VL, (s)dAb, Fab' and "r IgG" ("half antibodies").

[0060] The term "antibody construct" is understood to refer to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g. a full-length or complete immunoglobulin molecule, and / or is derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. The antibody construct is thus capable of binding to its specific target or antigen. Antibody constructs also include modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "multibodies" such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies, which comprise only one variable domain which may be a VHH, VH or VL that specifically binds to an antigen or epitope independent of other V regions or domains.

[0061] As used herein, the term "single-chain Fv", "single-chain antibody" or "scFv" is understood to refer to a single polypeptide chain antibody fragment that contains the variable regions from both the heavy and light chains but lacks the constant region. Usually, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that allows them to form the desired structure that allows antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods of making single chain antibodies are known, including those described in U.S. Patent Nos. 4,694,778 and 5,260,203; WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In specific embodiments, single chain antibodies can also be bispecific human and / or humanized and / or synthetic antibodies.

[0062] In certain embodiments, the first and second domains of the bispecific antibody are "bispecific single chain antibody constructs", more preferably bispecific "single chain Fvs" (scFvs). Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, as described herein, that allows their production, using recombinant techniques, as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same way as complete or full-length antibodies. Thus, scFvs are fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, usually linked by a short linker peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can either link the N-terminus of the VH to the C-terminus of the VL or vice versa. This scFv retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker.

[0063] Bispecific single chain variable fragments (bi-scFv or di-scFv with format (scFv)2) can be engineered by linking two scFv molecules (e.g., using a linker as described herein). Linking can be done by creating a single peptide chain with two VH and two VL regions to generate a tandem scFv (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules with a linker peptide that is too short (e.g., about 5 amino acids) to fold the two variable regions together, allowing the scFvs to dimerize. This type is known as a diabody (see, for example, Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0064] In certain embodiments, the first and second domains of the bispecific antibody specifically bind to a target cell surface antigen and human CD3, respectively. In certain embodiments, the first and second domains of the bispecific antibody form a bispecific antibody construct in a format selected from the group consisting of (scFv)2, scFv-single domain mAb, diabody, and any format of oligomer.

[0065] In certain embodiments, either the first, second or the first and second domains may comprise a single domain antibody, respectively a variable domain or at least the CDRs of a single domain antibody. Single domain antibodies contain only one (monomeric) antibody variable domain that is capable of selectively binding to a specific antigen, independent of other V regions or domains. The first single domain antibodies were made from heavy chain antibodies found in camels, and these are called VHH fragments. Cartilaginous fishes have also been derived from VHH fragments. NARWith heavy chain antibodies (IgNAR) from which single domain antibodies called fragments can be obtained. An alternative approach is to split the dimeric variable domain from a common immunoglobulin, for example from human or rodent origin, into monomers, thereby obtaining VH or VL as single domain Abs. Most research on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes. Examples of single domain antibodies are called sdAbs, nanobodies or single variable domain antibodies.

[0066] In certain embodiments, the first (binding) domain of the bispecific antibody binds to a target cell surface antigen. In some embodiments, the target cell surface antigen is CD70. CD70 (also known as CD27L or TNFSF7) is a type II integral membrane protein whose normal expression is restricted to a subset of activated T and B cells, mature dendritic cells, and medullary thymic epithelial cells.

[0067] In other embodiments, the target cell surface antigen is a tumor antigen. As used herein, the term "tumor antigen" is understood to refer to those antigens presented on tumor cells. These antigens can be presented on the cell surface with an extracellular portion, often combining the transmembrane and cytoplasmic portions of the molecule. These antigens can only be presented by tumor cells, never by normal cells. Tumor antigens can be expressed exclusively on tumor cells or can show tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More common antigens are antigens presented by tumor cells and normal cells, which are called tumor-associated antigens. These tumor-associated antigens can be overexpressed compared to normal cells, or are accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. In some embodiments, the second (binding) domain binds to a tumor antigen selected from CD19, CD33, epidermal growth factor receptor variant iii (EGFRvIII), mesothelin (MSLN), cadherin 19 (CDH19), FMS-like tyrosine kinase 3 (FLT3), delta-like ligand 3 (DLL3), placental cadherin (CDH3), B-cell maturation antigen (BCMA) or prostate-specific membrane antigen (PSMA). In some embodiments, the tumor antigen is a human tumor antigen.

[0068] In certain embodiments, the second (binding) domain of the bispecific antibody binds to epsilon of human CD3 on the surface of a T cell. In certain preferred embodiments, the second domain of the bispecific antibody binds to an extracellular epitope of the human CD3 epsilon chain. In some preferred embodiments, the second domain of the bispecific antibody that binds to an extracellular epitope of human CD3 comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the following: (a) CDR-L1 as set forth in SEQ ID NO: 27 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 28 of WO 2008 / 119567 and CDR-L3 as set forth in SEQ ID NO: 29 of WO 2008 / 119567; (b) CDR-L1 as set forth in SEQ ID NO: 117 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 118 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 119 of WO 2008 / 119567; and (c) CDR-L1 as set forth in SEQ ID NO: 153 of WO 2008 / 119567, CDR-L2 as set forth in SEQ ID NO: 154 of WO 2008 / 119567, and CDR-L3 as set forth in SEQ ID NO: 155 of WO 2008 / 119567.

[0069] In another preferred embodiment, the second domain of the bispecific antibody comprises a VH region which binds to an extracellular epitope of the human CD3 epsilon chain and comprises a CDR-H1, CDR-H2 and CDR-H3 selected from: (a) CDR-H1 as set forth in SEQ ID NO: 12 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 13 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 14 of WO 2008 / 119567; (b) CDR-H1 as set forth in SEQ ID NO: 30 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 31 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 32 of WO 2008 / 119567; (c) CDR-H1 as set forth in SEQ ID NO: 48 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 49 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 50 of WO 2008 / 119567; (d) CDR-H1 as set forth in SEQ ID NO: 66 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 67 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 68 of WO 2008 / 119567; (e) CDR-H1 as set forth in SEQ ID NO: 84 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 85 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 86 of WO 2008 / 119567; (f) CDR-H1 as set forth in SEQ ID NO: 102 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 103 of WO 2008 / 119567 and CDR-H3 as set forth in SEQ ID NO: 104 of WO 2008 / 119567; (g) CDR-H1 as set forth in SEQ ID NO: 120 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 121 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 122 of WO 2008 / 119567; (h) CDR-H1 as set forth in SEQ ID NO: 138 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 139 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 140 of WO 2008 / 119567; (i) CDR-H1 as set forth in SEQ ID NO: 156 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 157 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 158 of WO 2008 / 119567; and (j) CDR-H1 as set forth in SEQ ID NO: 174 of WO 2008 / 119567, CDR-H2 as set forth in SEQ ID NO: 175 of WO 2008 / 119567, and CDR-H3 as set forth in SEQ ID NO: 176 of WO 2008 / 119567.

[0070] In certain preferred embodiments, the above three groups of VL CDRs are combined with the above ten groups of VH CDRs in the second binding domain to form a group (30) comprising CDRs 1-3, respectively.

[0071] It is also preferred that the second domain that binds to CD3 comprises a VH region selected from the group of VH regions as set out in SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008 / 119567, or as set out in the present sequence listing as SEQ ID NO: 15 or 24.

[0072] It is preferred that the second domain which binds to CD3 comprises a VL region selected from the group of VL regions as set out in SEQ ID NO: 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179 or 183 of WO 2008 / 119567, or as set out in the present sequence listing as SEQ ID NO: 16 or 25.

[0073] More preferably, the bispecific antibody is characterized by a second domain that binds CD3 comprising a VL region and a VH region selected from: (a) a VL region as set forth in SEQ ID NO: 17 or 21 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 15 or 19 of WO 2008 / 119567; (b) a VL region as set forth in SEQ ID NO: 35 or 39 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 33 or 37 of WO 2008 / 119567; (c) a VL region as set forth in SEQ ID NO: 53 or 57 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 51 or 55 of WO 2008 / 119567; (d) a VL region as set forth in SEQ ID NO: 71 or 75 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 69 or 73 of WO 2008 / 119567; (e) a VL region as set forth in SEQ ID NO: 89 or 93 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 87 or 91 of WO 2008 / 119567; (f) a VL region as set forth in SEQ ID NO: 107 or 111 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 105 or 109 of WO 2008 / 119567; (g) a VL region as set forth in SEQ ID NO: 125 or 129 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 123 or 127 of WO 2008 / 119567; (h) a VL region as set forth in SEQ ID NO: 143 or 147 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 141 or 145 of WO 2008 / 119567; (i) a VL region as set forth in SEQ ID NO: 161 or 165 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 159 or 163 of WO 2008 / 119567; or (j) a VL region as set forth in SEQ ID NO: 179 or 183 of WO 2008 / 119567 and a VH region as set forth in SEQ ID NO: 177 or 181 of WO 2008 / 119567.

[0074] Also in a preferred embodiment, the bispecific antibody comprises a second domain that binds to CD3 comprising a VL region as set forth in SEQ ID NO: 16 or 25 and a VH region as set forth in the present sequence listing as SEQ ID NO: 15 or 24.

[0075] A preferred embodiment of said bispecific antibody is characterized by a second domain that binds to CD3 comprising an amino acid sequence selected from SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 of WO 2008 / 119567 or as set forth in the present sequence listing as SEQ ID NO: 26.

[0076] According to a preferred embodiment, the first and / or second domain has the following format: the pair of VH and VL domains is in the format of a single chain antibody (scFv). The VH and VL domains are arranged in the order VH-VL or VL-VH. It is preferred that the VH domain is arranged N-terminally to the linker sequence and the VL domain is arranged C-terminally to the linker sequence. In certain embodiments, the first and second domains of the bispecific antibody form a bispecific antibody in a format selected from (scFv)2, scFv-single domain mAb, diabody or oligomer in any of these formats.

[0077] In certain preferred embodiments, the bispecific antibody further comprises a third domain. In certain embodiments, the third domain is a single chain Fc (scFc) domain. In certain preferred embodiments, the scFc domain is an scFc half-life extension (HLE) domain.

[0078] An "Fc" portion or an "Fc" monomer is understood to refer to a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having the function of a CH3 domain of an immunoglobulin molecule. A polypeptide comprising this CH domain is a "polypeptide monomer". An Fc monomer may be a polypeptide comprising a fragment of an immunoglobulin constant region, excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but maintaining at least a functional portion of one CH2 domain and one functional portion of one CH3 domain, the CH2 domain being amino-terminal to the CH3 domain. In a preferred embodiment, an Fc monomer may be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region and a CH3 region, the hinge region being amino-terminal to the CH2 domain. The hinge region of a bispecific antibody is believed to promote dimerization. Such an Fc polypeptide molecule may be obtained, for example, by papain digestion of an immunoglobulin region (obtaining, of course, a dimer of two Fc polypeptides). In another embodiment, the Fc monomer can be a polypeptide region comprising a CH2 region and a portion of the CH3 region. Such Fc polypeptide molecules can be obtained, for example, by pepsin digestion of an immunoglobulin molecule. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the following Fc polypeptide sequences: IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region. (See, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). In one embodiment, the Fc monomer has an amino acid sequence as disclosed in WO 2014 / 153063. Because there is some variation between immunoglobulins, and simply for clarity, an Fc monomer is understood to refer to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM. As mentioned, the Fc monomer may also include a flexible hinge N-terminal to these domains.In the case of IgA and IgM, the Fc monomer may comprise a J chain. In the case of IgG, the Fc portion comprises the immunoglobulin domains CH2 and CH3 and the hinge between the first two domains and CH2. Although the boundaries of the Fc portion may vary, an example for a human IgG heavy chain Fc portion comprising functional hinge, CH2 and CH3 domains may be defined as comprising, for example, residues D231 (corresponding to D234 in Table 1 below - of the hinge domain) to P476, respectively L476 (for IgG4), at the carboxyl terminus of the CH3 domain, numbering according to Kabat. Two Fc portions or Fc monomers fused to each other via a peptide linker define the third domain of the antibody construct of the invention, which may also be defined as a scFc domain.

[0079] The IgG hinge region may be identified by similarity using the Kabat numbering as shown in Table 1. It is envisaged that the hinge domain / region of the third domain comprises amino acid residues corresponding to the stretch of IgG sequence from D234 to P243 according to the Kabat numbering. It is also envisaged that the hinge domain / region of the third domain comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 191) (corresponding to the stretch D234 to P243 as shown in Table 1 below - variations in the sequence are envisaged, provided that the hinge region still promotes dimerization). In a preferred embodiment, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the antibody construct is removed by a N314X substitution, where X is any amino acid except Q. This substitution is preferably a N314G substitution. In a more preferred embodiment, this CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0080] [Table 1]

[0081] In some embodiments, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 192), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 193) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 194) and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 195). The IgG1 subtype hinge sequence may be the following sequence EPKSCDKTHTCPPCP (as shown in Table 1 and SEQ ID NO: 196). Thus, these core hinge regions are also envisaged in the context of bispecific antibodies.

[0082] The locations and sequences of the IgG CH2 and IgG CD3 domains can be identified by similarity using the Kabat numbering as set forth in Table 2.

[0083] [Table 2]

[0084] In one embodiment, the amino acid residues highlighted in bold in the CH3 domain of the first or both Fc monomers are deleted.

[0085] When a linker is used to fuse the first domain with the second domain, or the first or second domain with the third domain, this linker is preferably of sufficient length and sequence to ensure that each of the first and second domains retains their different binding specificities independently of each other. In the case of peptide linkers connecting at least two binding domains (or two variable domains) in a bispecific antibody construct, these peptide linkers preferably contain only a few amino acid residues, for example 12 amino acid residues or less. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. Contemplated peptide linkers of less than 5 amino acids contain 4, 3, 2 or 1 amino acid, where Gly-rich linkers are preferred.

[0086] A particularly preferred "single" amino acid "peptide linker" is Gly. Thus, the peptide linker may consist of a single amino acid Gly. In a preferred embodiment, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 197), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 1 (e.g., 2 or 3). In another preferred embodiment, the peptide linker has the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 197), or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 5 (e.g., 5, 6, 7, 8, etc. or more). In certain embodiments, it is preferred that x is 6 ((Gly4Ser)6). Characteristics of peptide linkers, including the lack of promotion of secondary structure, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Peptide linkers that do not promote any secondary structure are preferred. Methods for preparing fused, operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g. WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0087] A preferred embodiment of the peptide linker for the fusion of the first and second domains has the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 197). A preferred linker embodiment of the peptide linker for the fusion of the second and third domains is the (Gly)4-linker and the G4-linker, respectively.

[0088] The peptide linker by which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomer") are fused to each other preferably comprises at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). It is also preferred that the linker comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, most preferably exactly 30 amino acid residues. Another embodiment of such a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 197), or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer equal to or greater than 5, e.g. 6, 7 or 8. Preferably, the integer is 6 or 7, more preferably, the integer is 6.

[0089] In some preferred embodiments, the third domain of the bispecific antibody comprises, in amino to carboxyl order: This is an HLE domain with the structure hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0090] In one certain embodiment, the CH2 domain of one or preferably each (both) polypeptide monomer of the third domain comprises an intradomain cysteine ​​disulfide bridge. As known in the art, the term "cysteine ​​disulfide bridge" refers to a functional group with the general structure RSSR. This linkage, also called SS bond or disulfide bridge, is obtained by coupling of two thiol groups of cysteine ​​residues. For bispecific antibodies, it is particularly preferred that the cysteines forming the cysteine ​​disulfide bridge in the mature antibody construct are introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering).

[0091] In one embodiment, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of the glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid except Q. The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0092] It is believed that the favourable properties of the bispecific antibody constructs, e.g. compared to bispecific hetero-Fc antibody constructs known in the art, may be linked, inter alia, to the introduction of the above-mentioned modifications in the CH2 domain. It is therefore preferred that the CH2 domain in the third domain of the bispecific antibody comprises intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321 and / or the glycosylation site at Kabat position 314 is removed by an N314X substitution, preferably an N314G substitution, as described above.

[0093] In a further preferred embodiment, the CH2 domain in the third domain of the bispecific antibody contains intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321 and the glycosylation site at Kabat position 314 is eliminated by a N314G substitution.

[0094] In certain embodiments, the third domain of the bispecific antibody comprises or consists of, in amino to carboxyl order: DKTHTCPPCP (SEQ ID NO: 191) (i.e. hinge)-CH2-CH3-linker-DKTHTCPPCP (SEQ ID NO: 191) (i.e. hinge)-CH2-CH3. The peptide linker of the above-mentioned bispecific antibody is in a preferred embodiment characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly4Ser (SEQ ID NO: 197 or a polymer thereof, i.e. (Gly4Ser)x, where x is an integer equal to or greater than 5 (e.g. 5, 6, 7, 8 etc. or more), with 6 being preferred ((Gly4Ser)6). The antibody may further comprise the substitution N314X as described above, preferably N314G and / or the further substitutions V321C and R309C.

[0095] Bispecific antibodies may also contain additional domains, e.g., useful for the isolation of the molecule or relevant for adapting the pharmacokinetic profile of the molecule. Domains useful for the isolation of the antibody construct may be selected from peptide motifs or secondarily introduced moieties that can be captured in an isolation method, e.g., a separation column. Non-limiting examples of such additional domains include peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding protein (MBP tags), Flag tags, Strep tags and variants thereof (e.g., StrepII tags) and His tags. All of the bispecific antibodies disclosed herein may contain a His tag domain, commonly known as a repeat of consecutive His residues, preferably five, more preferably six His residues (hexahistidine) in the amino acid sequence of the molecule. The His tag may, e.g., be located at either the N-terminus or the C-terminus of the antibody construct, but is preferably located at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 198) is linked to the C-terminus of the bispecific antibody via a peptide bond. In addition, the PLGA-PEG-PLGA conjugate system can be combined with a polyhistidine tag for sustained release applications and improved pharmacokinetic profiles.

[0096] In certain embodiments, a bispecific antibody comprises a first domain and a second domain: (i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains; (ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains; (iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or (iv) the first domain comprises one antibody variable domain and the second domain comprises one antibody variable domain.

[0097] Thus, the first and second domains may each be binding domains comprising two antibody variable domains, such as a VH and a VL domain. Examples for such binding domains comprising two antibody variable domains have been described above and include, for example, the Fv fragment, scFv fragment or Fab fragment described herein above. Alternatively, either or both of the binding domains may comprise only a single variable domain. Examples for such single domain binding domains have been described herein above and include, for example, nanobodies or single variable domain antibodies comprising only one variable domain, which may be a VHH, VH or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0098] In some preferred embodiments, the bispecific antibody comprises a first domain, a second domain and a third domain, the first domain binds CD70, the second domain binds human CD3 and the third domain is an HLE domain having, from amino to carboxyl: hinge-CH2-CH3-linker-hinge-CH2-CH3. In other preferred embodiments, the bispecific antibody comprises a first domain, a second domain and a third domain, the first domain binds a tumor antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, BCMA or PSMA, the second domain binds human CD3 and the third domain is an HLE domain having, from amino to carboxyl: hinge-CH2-CH3-linker-hinge-CH2-CH3. In preferred embodiments, the first and second domains are fused to the third domain via a peptide linker. Preferred peptide linkers are described herein above and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser, or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 1 (e.g., 2, 3, 4, 5, 6, or 7).

[0099] In some embodiments, the bispecific antibody is characterized in having an amino acid sequence selected from:

[0100] [Table 3]

[0101] Any of the above bispecific antibodies may or may not be provided with a third domain, which is a half-life extension (HLE) domain, preferably an scFc domain or a heteroFc domain or an albumin binding domain. The second domain of a bispecific antibody that binds to human CD3 may be connected to the N-terminus or C-terminus of the HLE domain (e.g. via a linker as described above).

[0102] In some embodiments, the bispecific antibody is a CD70xCD3 bispecific antibody, which comprises a first domain that binds CD70 and a second domain that binds CD3. In one embodiment, the first domain binds CD70 and has CDRs as set forth in SEQ ID NOs: 182-187, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 188, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 189. In one embodiment, the CD70xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 190.

[0103] In some embodiments, the bispecific antibody is a CD19xCD3 bispecific antibody, which comprises a first domain that binds to CD19 and a second domain that binds to CD3. In one embodiment, the first domain binds to CD19 and has CDRs as set forth in SEQ ID NOs: 1-6, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In another embodiment, the first domain binds to CD19 and has CDRs as set forth in SEQ ID NOs: 105-107 and 109-111, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14, and further comprises an HLE domain (third domain). In one embodiment, the CD19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17. In another embodiment, the CD19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 114.

[0104] In some embodiments, the bispecific antibody is a BCMAxCD3 bispecific antibody, which comprises a first domain that binds BCMA and a second domain that binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds BCMA and has CDRs as set forth in SEQ ID NOs: 126-131, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the BCMAxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 132, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 133. In one embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 135. In another embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 136.

[0105] In some embodiments, the bispecific antibody is a CD33xCD3 bispecific antibody, which comprises a first domain that binds CD33 and a second domain that binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds CD33 and has CDRs as set forth in SEQ ID NOs: 29-31 and 34-36, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the CD33xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 27 or 28, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32 or 33. In one embodiment, the CD33xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 40. In another embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:41.

[0106] In some embodiments, the bispecific antibody is an EGFRvIIIxCD3 bispecific antibody, which comprises a first domain that binds EGFRvIII and a second domain that binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds EGFRvIII and has CDRs as set forth in SEQ ID NOs: 42-47, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the EGFRvIIIxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 48, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 49. In one embodiment, the EGFRvIIIxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 52.

[0107] In some embodiments, the bispecific antibody is a MSLNxCD3 bispecific antibody, which comprises a first domain that binds to MSLN and a second domain that binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to MSLN and has CDRs as set forth in SEQ ID NOs: 53-58, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the MSLNxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 59, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 60. In one embodiment, the MSLNxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 63. In one embodiment, the MSLNxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 64.

[0108] In some embodiments, the bispecific antibody is a CDH19xCD3 bispecific antibody and comprises a first domain that binds to CDH19 and a second domain that binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to CDH19 and has CDRs as set forth in SEQ ID NOs: 65-70, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the CDH19xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 71, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 72. In one embodiment, the CDH19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 74-82. In one embodiment, the CDH19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:82.

[0109] In some embodiments, the bispecific antibody is a DLL3xCD3 bispecific antibody, which comprises a first domain that binds to DLL3 and a second domain that binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to DLL3 and has CDRs as set forth in SEQ ID NOs: 94-99, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the DLL3xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 100, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 101. In one embodiment, the DLL3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 104.

[0110] In some embodiments, the bispecific antibody is a FLT3xCD3 bispecific antibody, which comprises a first domain that binds FLT3 and a second domain that binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds FLT3 and has CDRs as set forth in SEQ ID NOs: 83-88, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the FLT3xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 89, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 90. In one embodiment, the FTL3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 93.

[0111] In some embodiments, the bispecific antibody is a CDH3xCD3 bispecific antibody, which comprises a first domain that binds to CDH3 and a second domain that binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to CDH3 and has CDRs as set forth in SEQ ID NOs: 115-120, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the CDH3xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 121, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 122. In one embodiment, the CDH3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 125.

[0112] In some embodiments, the bispecific antibody is a PSMAxCD3 bispecific antibody, which comprises a first domain that binds PSMA and a second domain that binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds PSMA and has CDRs as set forth in SEQ ID NOs: 137-142, and the second domain binds CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the PSMAxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 143, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 144. In one embodiment, the PSMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 146-151, 161-168, and 176-181. In one embodiment, the PSMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:177.

[0113] The bispecific antibodies disclosed herein can be prepared by methods known in the art. For example, the bispecific antibodies can be prepared by the methods disclosed in WO2008 / 119657 and WO2017 / 134140.

[0114] In some embodiments, the bispecific antibody is a masked bispecific antigen binding protein. Masked bispecific antigen binding proteins have been described previously. See, for example, WO 2017 / 040344; US 2015 / 0079088. A "masked bispecific antigen binding protein" or "masked bispecific binding protein" is understood to refer to a masked antigen binding protein that binds to two different antigens or epitopes. A "masked antigen binding protein" is a protein that comprises a masking domain (MD) coupled (e.g., through a covalent bond or linker) to an antigen binding domain (AB) such that coupling of the masking domain (MD) inhibits or reduces binding of the AB to its antigen. The MD further comprises a protein recognition site (PR) that comprises a substrate or binding site for a protein or protease such that when the protein or protease binds to and / or cleaves the protein recognition site, the AB domain binds to the antigen or binding to the antigen by the AB domain is increased or induced. Masked antigen binding proteins have been previously described, for example, in WO 2017 / 040344, U.S. Pat. No. 9,540,440, U.S. Pat. Appln. Pub. No. 20150118254, U.S. Pat. No. 9,127,053, U.S. Pat. No. 9,517,276, and U.S. Pat. No. 8,563,269. It will be apparent to one of skill in the art that in some embodiments, the masked antigen binding protein may lack an MD (e.g., when the MD is not linked to the masked antigen binding protein by a covalent bond (e.g., a disulfide bond between cysteine ​​residues)) since the PR is cleaved by a protease, thereby releasing at least the MD.

[0115] In some embodiments, the masked antigen binding protein is a probody (e.g., as described in Polu KR and Lowman HB. Expert Opin Biol Ther. 2014 Aug;14(8):1049-53; or Desnoyers LR et.al., Sci Transl Med. 2013 Oct 16;5(207)) or a ProTIA prodrug (e.g., as described in Schellenberger V. Amunix unveils next-generation immuno-oncologic cancer therapy platform. https: / / biopharmadealmakers.nature.com. September 2016 edition, B20; see also http: / / www.amunix.com / technology / pro-tia / ). In some embodiments, the masked antigen binding protein has the following structural arrangement from N-terminus to C-terminus: MD-AB or AB-MD. In some embodiments, the masked antigen binding protein comprises a connecting peptide (LP), and the masked antigen binding protein has the following structural arrangement from N-terminus to C-terminus: MD-LP-AB or AB-LP-MD.

[0116] When the masked antigen binding protein is in the presence of antigen in a naive state, binding of the AB to the antigen is reduced or inhibited when compared to binding of the AB to the antigen when the masked antigen binding protein is in an active state (i.e., when a protein or protease binds to the PR and / or removes or transfers the MD). When compared to binding of the AB not associated with the MD (i.e., when the masked antigen binding protein is in an active state), the antigen binding ability of the masked antigen binding protein in the naive state is reduced, for example, by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100%, as measured in in vitro and / or in vivo binding assays.

[0117] In some embodiments, the protein recognition site (PR) serves as a substrate for a protease, preferably an extracellular protease. The PR may be selected based on proteins or proteases produced by cells (including, for example, tumor cells) near the cells expressing the antigen and / or produced by cells (including, for example, tumor cells) that co-exist in tissues with the desired antigen of the AB of the masked antigen binding protein. In some embodiments, the protease is u-type plasminogen activator (uPA, also called urokinase), legumain and / or matriptase (also called MT-SP1 or MTSP1). In some embodiments, the protease is a matrix metalloprotease (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N. and Salvesen, G. Handbook of Proteolytic Enzymes (Third Edition). Elsevier, 2013. ISBN: 978-0-12-382219-2).

[0118] In some embodiments, one antibody or antigen-binding fragment thereof (AB1) domain of the bispecific antigen-binding protein has specificity for one target antigen and another antibody or antigen-binding fragment thereof (AB2) domain has specificity for another target antigen. In some embodiments, one antibody or antigen-binding fragment thereof (AB1) domain of the bispecific antigen-binding protein has specificity for one epitope of a target antigen and another antibody or antigen-binding fragment thereof (AB2) domain has specificity for another epitope of the same target antigen.

[0119] The terms "antibody fragment", "antibody fragment thereof" or "antigen-binding antibody fragment" are understood to refer to a portion of an intact antibody. An "antigen-binding fragment" or "antigen-binding fragment thereof" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigenic determining variable regions of an intact antibody. Examples of antigen-binding fragments of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv and single chain antibodies.

[0120] A masked bispecific antigen binding protein comprises at least one masking domain (MD) that comprises a protein recognition site (PR), which inhibits or reduces binding of the AB to an antigen. The at least one MD comprises a protein recognition site (PR) that comprises a substrate or binding site for a protein or protease, such that when the protein or protease binds to and / or cleaves the protease recognition site, the AB domain binds to the antigen or binding is increased. With respect to a bispecific masked antigen binding protein, the masked antigen binding protein preferably comprises two MDs (e.g., MD1 and MD2) that reduce the ability of each antigen binding domain (AB1 and AB2) to bind to its respective antigen or epitope.

[0121] The masked bispecific masked antigen binding proteins provided herein are stable in the circulation and are activated at the site of therapeutic and / or diagnostic intent, but not in normal (i.e., healthy) tissues. When in the active state, the masked antigen binding proteins and / or masked bispecific or multispecific antigen binding proteins exhibit binding to antigen that is at least equivalent to that of the corresponding unmodified antibody or bispecific or multispecific antigen binding protein (i.e., the equivalent of the antibody without the masking domain).

[0122] The masked bispecific antigen binding proteins described herein, in various embodiments, bind to human CD3. For example, in various aspects, the masked bispecific antigen binding proteins activate T cells through engagement of CD3ε on the T cells. That is, the antibodies agonize, stimulate, activate, and / or enhance CD3-mediated T cell activation. Biological activities of CD3 include, for example, T cell activation and other signaling through the interaction of CD3 with the antigen-binding subunit of the T cell receptor (TCR).

[0123] The masked bispecific antigen binding proteins disclosed herein optionally bind to CD3ε with a binding constant (Kd) ≦1 μM, e.g., in some embodiments ≦100 nM, ≦10 nM or ≦1 nM.

[0124] In various aspects, the masked bispecific antigen binding proteins bind to epidermal growth factor receptor (EGFR). The masked bispecific antigen binding proteins disclosed herein optionally bind to human EGFR with a binding constant (Kd) of ≦1 μM, e.g., in some embodiments, ≦100 nM, ≦10 nM, or ≦1 nM.

[0125] In a preferred embodiment, the masked bispecific or multispecific antigen binding protein binds to both CD3 and EGFR.

[0126] In some embodiments, the masked antigen binding protein is a heterodimer that comprises an antigen binding domain that is a Fab (e.g., an IgG Fab) and an antigen binding domain that is an scFv. For example, in a typical embodiment, the masked antigen binding protein comprises heavy and light chains (e.g., IgG heavy and light chains) that bind to one target (e.g., EGFR) and an scFv domain that binds to a second target (e.g., a T cell surface antigen such as CD3). Single chain antibodies (scFv) are antigen-binding proteins in which the VL and VH regions are linked to form a continuous protein chain via a linker (e.g., a synthetic sequence of amino acid residues, usually about 15 to about 20 amino acids in length), which is long enough to allow the protein chain to refold on itself and form a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). One example of a linker suitable for use in scFvs is GGGGSGGGSGGGGS (SEQ ID NO: 199). Other typical linkers contain at least 4 to 5 amino acids and range from about 4 residues to about 20 residues.

[0127] A typical format of a masked antigen-binding protein includes (i) two heavy chains comprising an scFv operably linked near the N-terminus of one or both heavy chains, and (ii) two light chains that associate with the heavy chains to form antigen-binding domains, where the scFv binds to CD3 and the Fab portion binds to EGFR. In this example, the antigen-binding protein includes three (or four) antigen-binding domains, which typically bind to two or more different antigens (or epitopes). One example of a linker suitable for linking the scFv to the heavy chain variable region is GGGGS (SEQ ID NO: 197). A typical linker contains at least one suitable linker having 4-5 amino acids.

[0128] A masked antigen binding protein comprises a masking domain (MD) coupled (e.g., by covalent or other form of attachment) to an antibody (AB). The masking domain (MD) comprises a masking peptide (or masking polypeptide) (MP) and a protein recognition site (PR). A masking peptide (or masking polypeptide) can be a stretch of amino acids that prevents binding of an antigen binding domain to its antigen. Generally, masking peptides, short sequences of 5-15 amino acids in length, are used, although shorter and longer sequences (i.e., masking polypeptides) are also contemplated. Masking domains are further described, for example, in WO 2017 / 040344; U.S. Patent Publication No. 2015 / 0079088 (incorporated by reference in its entirety, particularly with respect to its disclosure of masking domains for use with antibodies that bind EGFR) and U.S. Patent Publication No. 2016 / 0194399 (incorporated by reference in its entirety, particularly with respect to its disclosure of masking domains for use with antibodies that bind CD3).

[0129] In some embodiments, a masking peptide (or masking polypeptide) (MP) is attached to an antigen-binding domain (AB) via a protein recognition site (PR), which is optionally part of a larger linker sequence (i.e., a stretch of amino acids that links the MP to the antigen-binding protein). The PR serves as a substrate (or binding site) for a protein or protease, preferably an extracellular protease. The PR may be selected based on proteins or proteases produced by cells (including, for example, tumor cells) in the vicinity of cells expressing the target and / or produced by cells (including, for example, tumor cells) that coexist in a tissue with the desired target of at least one AB of the masked antigen-binding protein. In some embodiments, the protease is u-type plasminogen activator (uPA, also called urokinase), legumain, and / or matriptase (also called MT-SP1 or MTSP1). In some embodiments, the protease is a matrix metalloprotease (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N. and Salvesen, G. Handbook of Proteolytic Enzymes (Third Edition). Elsevier, 2013. ISBN: 978-0-12-382219-2. Alternatively, the MP is coupled to the antigen binding protein via a non-cleavable protein binding domain. In this regard, the PR is optionally an amino acid sequence that, upon interaction with a protein or protease, modulates the position of the MP and undergoes a conformational change such that the AB is free to bind to the target.

[0130] In various aspects of the disclosure, the masked bispecific antigen binding protein comprises a MD for each antigen binding portion of the construct (e.g., AB1, AB2, AB3, etc.). For example, a masked bispecific antigen binding protein comprising two Fab portions and two scFvs may comprise two sets of MDs, which may be the same or different independently. For example, the masked bispecific antigen binding protein comprises (i) two scFvs binding CD3 and MD1 linked to each scFv (optionally with the same MD for each scFv) and (ii) two Fab portions binding EGFR and MD2 linked to each Fab (optionally with the same MD for each Fab). In various aspects, the PRs of MD1 and MD2 comprise the same protein recognition sequence, allowing the MDs to be released for their respective binding regions in the same environment after association with a protease. The MDs may be attached at the N-terminus or C-terminus of the antigen binding domain, as long as the MDs can interfere with the binding of the antigen binding domain to the target and the linker does not prevent binding after the MDs are released. When the antigen binding domain is a Fab, the MD may be operably linked to the heavy chain variable region or the light chain variable region. In the example where the masked bispecific antigen binding protein comprises an intact antibody having both a Fab antigen binding domain and an scFv fused to a heavy chain (i.e. in a "stacked" conformation), the MD associated with the Fab antigen binding domain is preferably fused to the light chain variable region.

[0131] In some embodiments, one AB domain (e.g., AB1) in the masked bispecific antigen binding protein associates with a T cell surface ligand such as CD3, optionally in the form of an scFv. Exemplary masked antigen binding proteins that bind to CD3 include, but are not limited to, WO 2017 / 040344, US 20160194399.

[0132] In some embodiments, one AB domain (e.g., AB2) in the antigen binding protein associates with a tumor antigen, such as EGFR. Exemplary masked antigen binding proteins that associate with EGFR include, but are not limited to, the antibodies disclosed in US Patent Publication No. 20150079088.

[0133] In some embodiments, the masked antigen binding proteins (including, for example, masked bispecific antigen binding proteins) are modified to alter the isoelectric point of the antibody. For example, in some embodiments, one or more negatively charged, pH-sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are substituted with a positively charged amino acid (e.g., lysine or arginine). In some embodiments, the substitution of aspartic acid in one or more masking moieties of the antibody can increase the pI. In some embodiments, the one or more negatively charged, pH-sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are removed or substituted with a natural amino acid.

[0134] While masked antigen-binding proteins of the present disclosure (including, for example, bispecific or multispecific masked antigen-binding proteins) are often described herein as comprising intact antibody structures, the present disclosure also contemplates the use of antigen-binding antibody fragments that lack at least a portion of the traditional two heavy chain / two light chain structure. Fragments used as masked antigen-binding proteins comprise an antigen-binding domain (AB) operably linked to a masking domain (MD), as described above. For example, in some embodiments, the antigen-binding protein comprises two scFvs linked by a suitable linker (e.g., a stretch of amino acids long enough to allow each scFv to bind to its target). One or both scFvs are bound to the MD; if both scFvs are bound to the MD, the MDs can be the same or different (i.e., in various aspects, the MP and / or PR are different, but preferably the PR is the same).

[0135] The masked bispecific antibodies disclosed herein can be made by methods known in the art, such as those described in WO 2017 / 040344; U.S. Patent Application Publication No. 2015 / 0079088; and U.S. Patent Application Publication No. 2016 / 0194399.

[0136] Table 3 lists the sequences disclosed herein.

[0137] [Table 4]

[0138] [Table 5]

[0139] [Table 6]

[0140] [Table 7]

[0141] [Table 8]

[0142] [Table 9]

[0143] [Table 10]

[0144] [Table 11]

[0145]

Table 12

[0146]

Table 13

[0147]

Table 14

[0148]

Table 15

[0149]

Table 16

[0150]

Table 17

[0151]

Table 18

[0152]

Table 19

[0153]

Table 20

[0154]

Table 21

[0155]

Table 22

[0156]

Table 23

[0157]

Table 24

[0158]

Table 25

[0159]

Table 26

[0160]

Table 27

[0161]

Table 28

[0162]

Table 29

[0163]

Table 30

[0164]

Table 31

[0165]

Table 32

[0166] [Table 33]

[0167] [Table 34]

[0168] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. EXAMPLES

[0169] Example 1: Increased aggregation of BiTE molecules when stored under frozen conditions (-20°C) Compositions containing 1 mg / mL of each HLE BiTE (MSLNxCD3, CD19xCD3, CD33xCD3, BCMAxCD3, and DLL3xCD3) at pH 4.2 were filled into 5 mL vials and stored at -20°C for one month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates (e.g., bispecific antibody dimers) in the compositions. SE-UHPLC separates proteins in solution based on the hydrodynamic volume of the proteins using a size-exclusion ultrafast analytical column. High molecular weight (aggregate peak) elutes earlier than monomer and lower molecular weight peaks. The components were eluted isocratically and detected by UV detection, integrated, and the results are reported as the relative peak area % of the high molecular weight, main peak, and low molecular weight peaks. As shown in Figure 1, HMW aggregate levels increased in each composition after storage.

[0170] Example 2: pH and temperature dependence of BiTE molecular aggregation Compositions containing DLL3xCD3 HLE BiTE at a concentration of 1 mg / mL and pH 4.2, 4.8, or 6.3 were filled into 5 mL vials and stored for one month at -20° C. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the levels of HMW aggregates, the results of which are shown in Figure 2A. The HMW aggregates formed during storage appear to be pH dependent (Figure 2A).

[0171] Additionally, compositions containing DLL3xCD3 HLE BiTE at a concentration of 1 mg / mL were filled into 5 mL vials and stored at -10°C, -20°C, -30°C, -40°C, and -70°C for one month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the levels of HMW aggregates, the results of which are shown in Figure 2B. When BiTE was stored at -10°C, -20°C, and -30°C, the levels of HMW aggregates increased, and a slight increase in the levels of HMW aggregates was observed when BiTE was stored at temperatures lower than the Tg' of the composition containing BiTE (-32°C) (Figure 2B).

[0172] Example 3: Holding thawed BiTE at temperatures between 15°C and 30°C reduced aggregation levels Compositions containing DLL3xCD3 HLE BiTE at concentrations of 1 mg / mL, 5 mg / mL, and 13 mg / mL were filled into 5 mL vials and stored at -20°C for 12 months. After 12 months, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine levels of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. After the holding period, HMW aggregate levels had fallen to less than 5% (Figure 3A).

[0173] In a second set of experiments, compositions containing EGFRvIIIxCD3 BiTE at concentrations of 0.5 mg / mL and 2 mg / mL were filled into 5 mL vials and stored at -20°C for one month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the levels of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. HMW aggregate levels had fallen to less than 5% after the storage period (Figure 3B).

[0174] In a third series of experiments, compositions containing each of the HLE BiTEs (MSLNxCD3, CD19xCD3, CD33xCD3, CDH19xCD3, BCMAxCD3, DLL3xCD3, FLT3xCD3, PSMAxCD3, and CD70xCD3) at a concentration of 1 mg / mL were filled into 5 mL vials and stored at -20°C for one month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the levels of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. The levels of HMW aggregates in each composition had fallen to less than 1% after the storage period (Figure 3C).

[0175] In a fourth set of experiments, compositions containing each of the HLE BiTEs (CD33xCD3 and DLL3xCD3) at a concentration of 1 mg / mL were filled into 5 mL and stored at -30°C for 18 months. After 18 months, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the levels of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. The levels of HMW aggregates in each composition had fallen to less than 0.4% after the storage period (Figure 3D).

[0176] Example 4: Keeping thawed BiTE at temperatures between 15°C and 30°C does not affect BiTE stability Compositions containing DLL3xCD3 HLE BiTE at a concentration of 13 mg / mL were filled into 5 mL vials and stored at -20°C for 12 months. After 12 months, the compositions were thawed at room temperature and the thawed compositions were kept at room temperature for an additional 24 hours. Several stability analyses were performed after the 24 hour holding period and the results are shown in Table 4.

[0177] The potency of the HLE BiTE molecules was measured by a cell-based bioassay that measures cell death by loss of luminescence in carcinoma cell lines. The biological activity of the test samples is determined by comparing the response of the test samples to that of a reference standard (relative potency).

[0178] Clipping of BiTE molecules was measured using reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). rCE-SDS separates proteins based on their hydrodynamic size under reducing and denaturing conditions. Proteins were denatured, reduced, and injected into a bare fused silica capillary filled with a polymer gel matrix. A voltage was applied across the capillary and the SDS-coated proteins were separated based on their hydrodynamic size; smaller proteins migrate faster than larger proteins. Proteins were detected and integrated using a photodiode array (PDA) detector, and results are reported as the relative peak area % of the low molecular weight, main peak, and high molecular weight peak.

[0179] Cation exchange high performance liquid chromatography (CEX-HPLC) was used to measure the charge variants of BiTE molecules. A mobile phase gradient of increasing ionic strength at the appropriate pH was used to elute the protein charge variants. Proteins with less positive surface charge elute earlier than proteins with more positive surface charge. Eluted charge variants were detected by UV detection, integrated, and results for the main, acidic, and basic peaks were reported as % of the total peak area.

[0180] As shown in Table 4, storage of thawed BiTE did not negatively affect stability, although HMW levels decreased after the storage period.

[0181] [Table 35]

[0182] Example 5: Dependence of the decrease in HMW level of BiTE molecules after storage under frozen conditions on holding time and temperature A composition containing 1 mg / mL each of HLE BiTEs (CD33xCD3 and DLL3xCD3) and 2 mg / mL of BiTE (EGFRvIIIxCD3) was filled into 5 mL vials and stored for one month at -20° C. After one month, the composition was thawed at room temperature and immediately analyzed by size-exclusion ultra-performance liquid chromatography (SE-UHPLC) to determine the level of HMW aggregates (e.g., bispecific antibody dimers) in the composition.

[0183] The thawed compositions were held at various temperatures and HMW aggregate levels were analyzed by SE-UHPLC at different time points over a maximum holding time of 96 hours. The holding times required to reduce HMW aggregate levels to the initial pre-freeze levels for CD33xCD3 and DLL3xCD3 HLE BiTEs and EGFRvIIIxCD3 BiTEs at various temperatures were identical and are shown in FIG. 4.

[0184] Example 6: Stabilizing effect of benzyl alcohol (BA) Compositions containing 1 mg / mL of each of HLE BiTEs (CD19xCD3, CD33xCD3, BCMAxCD3, DLL3xCD3, and EGFRvIIIxCD3) were filled into 5 mL volumes and stored at -20°C for 4 weeks in the presence and absence of BAs. After 4 weeks, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine levels of HMW aggregates. The presence of BAs stabilizes BiTEs during storage (Figure 5).

[0185] Without being limited thereto, the present invention includes the following aspects. [Aspect 1] 1. A method for reducing aggregates of a bispecific antibody, comprising: 13. A method comprising maintaining the thawed bispecific antibody at a temperature between 5°C and 45°C for at least 4 hours, wherein the bispecific antibody was stored at a temperature between -20°C and -40°C prior to thawing. [Aspect 2] 2. The method of embodiment 1, wherein the bispecific antibody is a drug substance. [Aspect 3] 3. The method according to any one of the preceding claims, wherein the bispecific antibody is maintained at said temperature for a period of from 4 hours to 96 hours. [Aspect 4] 4. The method according to any one of aspects 1 to 3, wherein the bispecific antibody is maintained at a temperature of 10°C to 30°C for 8 hours to 48 hours. [Aspect 5] 5. The method according to any one of aspects 1 to 4, wherein the bispecific antibody is thawed at a temperature between 5°C and 45°C. [Aspect 6] The method according to any one of aspects 1 to 5, wherein the bispecific antibody has been stored at a temperature of -20°C to -35°C. [Aspect 7] 7. The method according to any one of aspects 1 to 6, wherein the bispecific antibody has been stored at about -30°C. [Aspect 8] 8. The method of any one of aspects 1-7, wherein the aggregates comprise high molecular weight (HMW) aggregates. [Aspect 9] 9. The method of any one of aspects 1 to 8, wherein said bispecific antibody contains less than about 1% of said HMW aggregates after said holding period. [Aspect 10] 10. The method of embodiment 9, wherein the bispecific antibody comprises less than about 0.5% of said HMW aggregates. [Aspect 11] 11. The method according to any one of aspects 1 to 10, wherein the HMW aggregate comprises a dimer of the bispecific antibody. [Aspect 12] 1. A method for preparing a composition comprising a bispecific antibody, comprising the steps of: Thawing the drug substance containing the bispecific antibody that has been stored at a temperature of -20°C to -40°C; maintaining the thawed drug substance containing the bispecific antibody at a temperature between 5° C. and 45° C. for at least 4 hours; A method comprising: [Aspect 13] 13. The method of embodiment 12, wherein the composition is a pharmaceutical composition comprising the bispecific antibody. [Aspect 14] 14. The method of any one of claims 12 to 13, further comprising filtering the drug substance. [Aspect 15] 15. The method of any one of aspects 12 to 14, further comprising dividing the composition into dosage forms. [Aspect 16] 16. The method according to any one of aspects 12 to 15, wherein the drug substance is held at said temperature for a period of from 4 hours to 96 hours. [Aspect 17] 17. The method according to any one of aspects 12 to 16, wherein the drug substance is maintained at a temperature of 10°C to 30°C for a period of 8 hours to 48 hours. [Aspect 18] 18. The method according to any one of aspects 12 to 17, wherein the drug substance is thawed at a temperature between 5°C and 45°C. [Aspect 19] 19. The method according to any one of aspects 12 to 18, wherein the drug substance has been stored at a temperature between -20°C and -35°C. [Aspect 20] 20. The method of any one of aspects 12-19, wherein the drug substance has been stored at about -30°C. [Aspect 21] 1. A method for preparing a composition comprising a bispecific antibody, the method comprising maintaining a thawed drug substance comprising a bispecific antibody at a temperature between 5° C. and 45° C. for at least 4 hours, wherein the drug substance was frozen at or above the glass transition temperature (Tg') of the drug substance prior to thawing. [Aspect 22] 22. The method of aspect 21, wherein the drug substance was frozen at a temperature between −10° C. and above the Tg′ of the drug substance prior to thawing. [Aspect 23] 23. The method of any one of claims 21 to 22, wherein the drug substance has been frozen at about -32°C. [Aspect 24] 24. The method according to any one of aspects 21 to 23, wherein the drug substance is held at said temperature for a period of from 4 hours to 96 hours. [Aspect 25] 25. The method according to any one of aspects 21 to 24, wherein the drug substance is thawed at a temperature between 5°C and 45°C. [Aspect 26] 26. The method according to any one of aspects 21 to 25, wherein said drug substance is maintained at the same temperature as the temperature at which said drug substance is thawed. [Aspect 27] 27. The method of claim 26, wherein the drug substance is thawed and maintained at a temperature between 15° C. and 30° C. for 30 hours to 50 hours. [Aspect 28] 28. The method of any one of aspects 21 to 27, further comprising dividing the composition into dosage forms. [Aspect 29] 29. The method of any one of aspects 12-28, wherein the drug substance contains less than about 1% HMW aggregates after the holding period. [Aspect 30] 30. The method of embodiment 29, wherein said drug substance comprises less than about 0.5% of said HMW aggregates. [Aspect 31] 31. The method of embodiment 29 or 30, wherein said HMW aggregates comprise dimers of said bispecific antibody. [Aspect 32] 32. The method according to any one of aspects 1 to 31, wherein the pharmaceutical substance comprises the bispecific antibody at a concentration of from about 0.05 mg / mL to about 20 mg / mL. [Aspect 33] 33. The method of any one of aspects 12 to 32, further comprising lyophilizing the composition. [Aspect 34] 33. The method of any one of aspects 12-32, further comprising spray drying the composition. [Aspect 35] 35. The method according to any one of aspects 1 to 34, wherein the bispecific antibody comprises: a first binding domain that binds to a target cell surface antigen; and a second binding domain that binds to human CD3, wherein the bispecific antibody is in an (scFv)2 format. [Aspect 36] 36. The method of embodiment 35, wherein the target cell surface antigen is CD19, CD33, or BCMA. [Aspect 37] 37. The method of embodiment 36, wherein the first binding domain comprises a VH domain and a VL domain; the VH comprises the amino acid sequence of SEQ ID NO: 77 and the VL comprises the amino acid sequence of SEQ ID NO: 78; the VH comprises the amino acid sequence of SEQ ID NO:28 and the VL comprises the amino acid sequence of SEQ ID NO:32 or 33; or A method wherein the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO: 133. [Aspect 38] 38. The method of embodiment 37, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 17, 40, or 135. [Aspect 39] 39. The method according to any one of aspects 1 to 38, wherein the bispecific antibody further comprises a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, and the two polypeptide monomers are linked to each other via a peptide linker. [Aspect 40] 40. The method of embodiment 39, wherein said third domain comprises, in amino to carboxyl order, hinge-CH2-CH3-linker-hinge-CH2-CH3. [Aspect 41] The method of embodiment 40, wherein the third domain is an HLE domain. [Aspect 42] 42. The method of any one of aspects 39 to 41, wherein the first binding domain binds to at least one target cell surface antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA, or PSMA. [Aspect 43] 43. The method of embodiment 42, wherein the first binding domain comprises a VH domain and a VL domain; the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 109; the VH comprises the amino acid sequence of SEQ ID NO:27 and the VL comprises the amino acid sequence of SEQ ID NO:32; the VH comprises the amino acid sequence of SEQ ID NO: 48 and the VL comprises the amino acid sequence of SEQ ID NO: 49; the VH comprises the amino acid sequence of SEQ ID NO:59 and the VL comprises the amino acid sequence of SEQ ID NO:60; the VH comprises the amino acid sequence of SEQ ID NO: 77 and the VL comprises the amino acid sequence of SEQ ID NO: 78; the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 112; the VH comprises the amino acid sequence of SEQ ID NO:89 and the VL comprises the amino acid sequence of SEQ ID NO:90; the VH comprises the amino acid sequence of SEQ ID NO: 100 and the VL comprises the amino acid sequence of SEQ ID NO: 101; the VH comprises the amino acid sequence of SEQ ID NO: 121 and the VL comprises the amino acid sequence of SEQ ID NO: 122; the VH comprises the amino acid sequence of SEQ ID NO: 188 and the VL comprises the amino acid sequence of SEQ ID NO: 189; the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO: 133; or A method wherein the VH comprises the amino acid sequence of SEQ ID NO: 173 and the VL comprises the amino acid sequence of SEQ ID NO: 174. [Aspect 44] 44. The method of embodiment 43, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 63, 114, 41, 82, 136, 104, 93, 177, 125, 190, or 52. [Aspect 45] 45. The method of embodiment 44, wherein the bispecific antibody consists of the amino acid sequence of SEQ ID NO: 63, 114, 41, 82, 136, 104, 93, 177, 125, 190, or 52. [Aspect 46] The method according to any one of aspects 1 to 34, wherein the bispecific antibody is a masked bispecific antigen-binding protein. [Aspect 47] 47. The method of embodiment 46, wherein the masked bispecific antigen-binding protein comprises: (a) a first antibody or antigen-binding fragment thereof (AB1) that binds to a first antigen and a masking domain (MD1) coupled to AB1, said MD1 comprising: (1) a first masking peptide (MP1) that inhibits or reduces binding of AB1 to its antigen; and (2) containing a protein recognition site (PR1); Binding to or cleavage of PR1 by a protein or protease increases AB1 binding to its antigen; a first antibody or antigen-binding fragment thereof (AB1) and a masking domain (MD1); (b) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen and a second masking domain (MD2) coupled to AB2, wherein the MD2 comprises: (1) a second masking peptide (MP) that inhibits or reduces binding of AB2 to its antigen; and (2) comprising a second protein recognition site (PR2); Binding to or cleavage of PR2 by a protein or protease increases AB2 binding to its antigen; a second antibody or antigen-binding fragment thereof (AB2) and a second masking domain (MD2); A method comprising: [Aspect 48] 48. The method of embodiment 46 or 47, wherein said PR1 and PR2 comprise the same protein recognition sequence. [Aspect 49] The method according to any one of aspects 46 to 48, wherein said AB1 binds to human CD3, and said AB2 binds to human EGFR. All references cited in this application are hereby incorporated by reference.

Claims

1. 1. A method for reducing aggregates of a bispecific antibody, comprising: maintaining the thawed bispecific antibody composition at a temperature between 5° C. and 45° C. for at least 4 hours; The bispecific antibody was stored at a temperature of −20° C. to −40° C. before thawing, and the bispecific antibody is (scFv ２ The format is and the composition has a pH of 3 to 7 and comprises the bispecific antibody and a buffer. The method.

2. 10. The method of claim 1, wherein the bispecific antibody is a drug substance.

3. The method of claim 1 or 2, wherein the bispecific antibody is maintained at said temperature for between 4 hours and 96 hours.

4. The method of any one of claims 1 to 3, wherein the bispecific antibody is maintained at a temperature between 10°C and 30°C for a period between 8 hours and 48 hours.

5. The method of any one of claims 1 to 4, wherein the bispecific antibody is thawed at a temperature between 5°C and 45°C.

6. The method of any one of claims 1 to 5, wherein the bispecific antibody has been stored at a temperature of -20°C to -35°C.

7. The method of any one of claims 1 to 6, wherein the bispecific antibody has been stored at -30°C.

8. The method of any one of claims 1 to 7, wherein the aggregates comprise high molecular weight (HMW) aggregates.

9. 9. The method of claim 8, wherein the bispecific antibody contains less than 1% of the HMW aggregates after the holding period.

10. 10. The method of claim 9, wherein the bispecific antibody contains less than 0.5% of the HMW aggregates.

11. The method of any one of claims 8 to 10, wherein the HMW aggregates comprise dimers of the bispecific antibody.

12. 1. A method for preparing a composition comprising a bispecific antibody, comprising the steps of: Stored at temperatures between -20°C and -40°C (scFv) ２ thawing a drug substance comprising a bispecific antibody of the following format; maintaining the thawed drug substance containing the bispecific antibody at a temperature between 5° C. and 45° C. for at least 4 hours; Including, wherein the composition has a pH between 3 and 7 and comprises the bispecific antibody and a buffer. method.

13. The method of claim 12, wherein the composition is a pharmaceutical composition comprising the bispecific antibody.

14. 14. The method of claim 12 or 13, further comprising filtering the drug substance.

15. The method of any one of claims 12 to 14, further comprising dividing the composition into dosage forms.

16. 16. The method of any one of claims 12 to 15, wherein the drug substance is held at said temperature for a period of from 4 hours to 96 hours.

17. 17. The method according to any one of claims 12 to 16, wherein the drug substance is maintained at a temperature of from 10°C to 30°C for a period of from 8 hours to 48 hours.

18. 18. The method according to any one of claims 12 to 17, wherein the drug substance is thawed at a temperature between 5°C and 45°C.

19. 19. The method of any one of claims 12 to 18, wherein the drug substance has been stored at a temperature of -20°C to -35°C.

20. 20. The method of any one of claims 12 to 19, wherein the drug substance has been stored at -30°C.

21. A method for preparing a composition comprising a bispecific antibody, comprising: ２ maintaining a thawed drug substance comprising a bispecific antibody of the formula (I) at a temperature between 5° C. and 45° C. for at least 4 hours, said drug substance having been frozen at or above the glass transition temperature (Tg′) of said drug substance prior to thawing. wherein the composition has a pH between 3 and 7 and comprises the bispecific antibody and a buffer. method.

22. 22. The method of claim 21, wherein the drug substance was frozen at a temperature between -10°C and above the Tg' of the drug substance prior to thawing.

23. 23. The method of claim 21 or 22, wherein the drug substance has been frozen at -32°C.

24. 24. The method of any one of claims 21 to 23, wherein the drug substance is held at said temperature for a period of from 4 hours to 96 hours.

25. 25. The method according to any one of claims 21 to 24, wherein the drug substance is thawed at a temperature between 5°C and 45°C.

26. 26. The method of any one of claims 21 to 25, wherein the drug substance is maintained at the same temperature at which it is thawed.

27. 27. The method of claim 26, wherein the drug substance is thawed and held at a temperature between 15°C and 30°C for 30 hours to 50 hours.

28. The method of any one of claims 21 to 27, further comprising dividing the composition into dosage forms.

29. 29. The method of any one of claims 12 to 28, wherein the drug substance contains less than 1% HMW aggregates after the holding period.

30. 30. The method of claim 29, wherein the drug substance contains less than 0.5% of the HMW aggregates.

31. 31. The method of claim 29 or 30, wherein the HMW aggregate comprises a dimer of the bispecific antibody.

32. 32. The method of any one of claims 1 to 31, wherein the drug substance comprises the bispecific antibody at a concentration of 0.05 mg / mL to 20 mg / mL.

33. The method of any one of claims 12 to 32, further comprising lyophilizing the composition.

34. The method of any one of claims 12 to 32, further comprising spray drying the composition.

35. The method according to any one of claims 1 to 34, wherein the bispecific antibody comprises: a first binding domain that binds to a target cell surface antigen; and a second binding domain that binds to human CD3. The method.

36. 36. The method of claim 35, wherein the target cell surface antigen is CD19, CD33 or BCMA.

37. 37. The method of claim 36, wherein the first binding domain comprises a VH domain and a VL domain, the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO:78; the VH comprises the amino acid sequence of SEQ ID NO:28 and the VL comprises the amino acid sequence of SEQ ID NO:32 or 33; or The method, wherein the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO:

133.

38. 38. The method of claim 37, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 17, 40 or 135.

39. 39. The method of any one of claims 1 to 38, wherein the bispecific antibody further comprises a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 and a CH3 domain, and wherein the two polypeptide monomers are linked together via a peptide linker.

40. 40. The method of claim 39, wherein the third domain comprises, in amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

41. 41. The method of claim 40, wherein the third domain is an HLE domain.

42. The method of any one of claims 35, 39-41, wherein the bispecific antibody has a first binding domain that binds to at least one target cell surface antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA, or PSMA.

43. 43. The method of claim 42, wherein the first binding domain comprises a VH domain and a VL domain, the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 109; the VH comprises the amino acid sequence of SEQ ID NO:27 and the VL comprises the amino acid sequence of SEQ ID NO:32; the VH comprises the amino acid sequence of SEQ ID NO:48 and the VL comprises the amino acid sequence of SEQ ID NO:49; the VH comprises the amino acid sequence of SEQ ID NO:59 and the VL comprises the amino acid sequence of SEQ ID NO:60; the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO:78; the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 112; the VH comprises the amino acid sequence of SEQ ID NO:89 and the VL comprises the amino acid sequence of SEQ ID NO:90; the VH comprises the amino acid sequence of SEQ ID NO: 100 and the VL comprises the amino acid sequence of SEQ ID NO: 101; the VH comprises the amino acid sequence of SEQ ID NO: 121 and the VL comprises the amino acid sequence of SEQ ID NO: 122; the VH comprises the amino acid sequence of SEQ ID NO: 188 and the VL comprises the amino acid sequence of SEQ ID NO: 189; the VH comprises the amino acid sequence of SEQ ID NO: 132 and the VL comprises the amino acid sequence of SEQ ID NO: 133; or A method wherein the VH comprises the amino acid sequence of SEQ ID NO: 173 and the VL comprises the amino acid sequence of SEQ ID NO:

174.

44. 44. The method of claim 43, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

45. 45. The method of claim 44, wherein the bispecific antibody consists of the amino acid sequence of SEQ ID NO: 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

46. The method of any one of claims 1 to 34, wherein the bispecific antibody is a masked bispecific antigen-binding protein.

47. 47. The method of claim 46, wherein the masked bispecific antigen binding protein comprises: (a) a first antibody or antigen-binding fragment thereof (AB1) that binds to a first antigen and a masking domain (MD1) coupled to AB1, wherein said MD1 comprises: (1) a first masking peptide (MP1) that inhibits or reduces binding of AB1 to its antigen; and (2) containing a protein recognition site (PR1); Binding to or cleavage of PR1 by a protein or protease increases AB1 binding to its antigen; a first antibody or antigen-binding fragment thereof (AB1) and a masking domain (MD1); (b) a second antibody or antigen-binding fragment thereof (AB2) that binds to a second antigen and a second masking domain (MD2) coupled to AB2, wherein the MD2 comprises: (1) a second masking peptide (MP) that inhibits or reduces binding of AB2 to its antigen; and (2) a second protein recognition site (PR2); Binding to or cleavage of PR2 by a protein or protease increases AB2 binding to its antigen; a second antibody or antigen-binding fragment thereof (AB2) and a second masking domain (MD2); A method comprising:

48. 48. The method of claim 47, wherein PR1 and PR2 contain the same protein recognition sequence.

49. The method of claim 47 or 48, wherein AB1 binds to human CD3 and AB2 binds to human EGFR.

50. The method of any one of claims 1 to 49, wherein the composition further comprises a sugar and a surfactant.

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