Methods for reducing aggregation of bispecific antibodies

Abstract

To provide methods for reducing aggregation of bispecific antibodies resulting from storage under frozen conditions.SOLUTION: Provided herein is, for example, a method for reducing aggregation of a bispecific T cell engager (BiTE) antibody construct resulting from storage under a frozen condition, by holding the antibody at a certain temperature after thaw.SELECTED DRAWING: None

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 particularly, to methods for reducing aggregation of bispecific antibodies disclosed herein, such as bispecific T cell engager antibodies, that occur as a result of storage under frozen conditions. [Background technology]

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

[0004] The freeze / thaw process can cause stress to proteins. For example, during the freezing process of a protein drug substance, water crystallizes, causing protein molecules to reach concentration levels several times higher than their initial levels. As a result of the concentration change, the thermodynamic stability of the protein can be compromised, leading to unfolding events and causing 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 present during the thawing process, which can be severe because 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 because they can adversely affect the protein's stability, immunogenicity, and efficacy. There is a need for methods 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 bispecific antibodies at a particular 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 a 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 before thawing, and in another embodiment, the bispecific antibody was stored at about -30°C before thawing. In one embodiment, the aggregates comprise high molecular weight (HMW) aggregates. In one embodiment, the HMW aggregates comprise bispecific antibody dimers. 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 the 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 was 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, disclosed herein is a method for preparing a composition comprising a bispecific antibody, the method comprising holding a thawed drug substance comprising the 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 was thawed. In another embodiment, the drug substance is thawed and held at the same temperature, 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 dosage forms. In another embodiment, the method for preparing the composition further comprises lyophilizing the composition. In 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 comprises 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, and the BiTE 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. 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, which are 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 has 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 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 peptide phosphorylation domains. The bispecific antibodies are linked to each other via a carrier and comprise or consist 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, 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 MD1 comprises (1) a first masking peptide (MP1) that inhibits or reduces binding of AB1 to the 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 the 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 to human CD3 and AB2 binds to human EGFR. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows the increase in aggregate levels (ΔHMW%) after 1 month of storage at -20°C for various HLE BiTE molecules. [Figure 2A] FIG. 2A shows the increase in aggregate levels (ΔHMW%) of DLL3xCD3 HLE BiTE after 1 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 1 month of storage at different temperatures. [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. [Figure 5] FIG. 5 shows the stabilizing effect of benzyl alcohol. DETAILED DESCRIPTION OF THE INVENTION

[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 terms "aggregate" or "aggregation" refer to the association of two or more molecules. In certain embodiments, aggregates are high molecular weight molecules having a larger molecular weight and / or size than non-aggregated molecules. 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 molecular size, 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 aggregates of a bispecific antibody is disclosed herein, the method 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 bispecific antibody-containing drug substance" is understood to refer to a bispecific antibody in a liquid state or a drug substance in a liquid state comprising a bispecific antibody that has been brought from a frozen state as a result of exposure to heat. In certain embodiments, a thawed bispecific antibody or a thawed bispecific antibody-containing drug substance is a bispecific antibody or a bispecific antibody-containing drug substance in a liquid state that is free or substantially free of frozen materials.

[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) so that it can be formulated into a pharmaceutical composition without further substantial purification and / or contaminant removal steps. The term drug substance encompasses compositions comprising a substantially purified recombinant protein (e.g., a bispecific antibody) and one or more pharmaceutically 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. Typical excipients include buffers (e.g., acetate buffer, glutamate buffer, citrate buffer, lactate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, or phosphate buffer), sugars (e.g., glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, or xylitol), and surfactants (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), buffer (e.g., glutamate buffer or citrate buffer), sugars (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, such as from about 0°C to about 50°C, or from about 0°C to about 40°C, or from about 0°C to about 30°C, or from about 5°C to about 45°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 40°C, or from about 10°C to about 30°C, or from about 15°C to about 50°C, or from about 15°C to about 40°C, or from about 15°C to about 30°C, or from about 20°C to about 30°C, or The bispecific antibody is thawed at a temperature of 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 will be appreciated by those skilled in the art, the bispecific antibody can be gently mixed during thawing to ensure a uniform temperature distribution and / or disruption of concentration gradients formed during thawing. Gentle mixing can be achieved, for example, by using a tilting shaker or by gently inverting the container of the bispecific antibody. Alternatively, the bispecific antibody can 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 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 5°C to about 40°C, or about 5°C to about 30°C, or about 10°C to about 50°C, or about 10°C to about 45°C, or about 10°C to about 40°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 maintaining temperature is 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 temperatures above 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, 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 48 hours, or about 15 hours to about 30°C. 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 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.

[0028] The bispecific antibodies were stored under frozen conditions before 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 above 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 above 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 above and below the stated value or range, including 20 percent, e.g., 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent.

[0031] As used herein, the term "storing" or "stored" 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 can be achieved, for example, by using a freezer, refrigerated truck, or shipping device capable of maintaining the required temperature. As will be recognized by one of skill in the art, the antibody is frozen when stored under the storage temperatures described above.

[0032] Bispecific antibodies can be stored in any suitable container that can maintain its integrity at storage temperatures. Typical containers include vials, bottles, bags, and carboys. Such containers are well known in the art and commercially available. In certain embodiments, bispecific antibodies are stored in disposable containers, such as commercially available flexible freeze-thaw containers, e.g., 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 It is about 12L.

[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 compositions comprising bispecific antibodies. In certain embodiments, the methods comprise thawing a bispecific antibody-containing drug substance that has been stored frozen and maintaining the thawed drug substance at a temperature for at least 4 hours.

[0035] In certain embodiments, the bispecific antibody drug substance has been stored at a temperature of about -20°C to about -50°C. In certain embodiments, the bispecific antibody drug substance has been stored at a temperature of about -20°C to about -35°C. In certain embodiments, the bispecific antibody drug substance has been stored at a temperature of about -25°C to about -35°C. In certain embodiments, the bispecific antibody 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 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, methods for preparing a composition comprising a bispecific antibody comprise maintaining a thawed drug substance comprising a bispecific antibody at a temperature for at least 4 hours, where the drug substance was 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 the Tg' of the drug substance (e.g., from about -10°C to above Tg') for about 1 day to about 5 years, or from 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 above 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 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 capable of maintaining its integrity under storage / freezing temperatures. Typical 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, e.g., 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 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, or 12 L.

[0040] A drug substance comprising a bispecific antibody can 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 methods comprise 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, or 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 10°C to about 45°C for 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. The solution is maintained at a temperature of about 15°C to about 40°C for about 4 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 bispecific antibody-containing drug substance 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 bispecific antibody-containing drug substance 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 a 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 bispecific antibody-containing drug substance is reduced to approximately the same level as before storage under frozen conditions. In certain embodiments, the thawed drug substance comprising a bispecific antibody is maintained at a temperature of about 5° C. to about 45° C. for a period of about 4 hours to the time at which the level of aggregates in the bispecific antibody-containing drug substance 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 a thawed drug substance to approximately the same level as before storage under frozen conditions at a particular temperature 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 maintained at the same temperature at which the drug substance is thawed. For example, a drug substance comprising a bispecific antibody can be thawed at any of the thawing and maintaining temperatures disclosed above, e.g., from about 5°C to about 45°C, and then maintained at the same temperature for at least 4 hours after thawing (e.g., from about 4 hours to about 150 hours, or from about 4 hours to about 120 hours, or from about 4 hours to about 96 hours, or from about 4 hours to about 72 hours, or from about 4 hours to about 48 hours, or from about 4 hours to about 24 hours, or from about 8 hours to about 96 hours, or from about 8 hours to about 72 hours, or from about 8 hours to about 48 hours, or from about 24 hours to about 72 hours, or from about 24 hours to about 48 hours). Whether a drug substance is thawed can be readily determined by one of ordinary skill in the art.

[0045] In certain embodiments, the drug substance is maintained at the same temperature at which the drug substance is thawed, and the drug substance is maintained 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 skilled in the art can determine the level of aggregates in the drug substance before freezing and at various time points after thawing using methods known in the art, such as SE-UHPLC. In one embodiment, the drug substance is thawed 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 held at the same temperature, from about 5°C to about 45°C, for a total period of from about 30 hours to about 100 hours, or from about 30 hours to about 90 hours, or from about 30 hours to about 80 hours, or from about 30 hours to about 70 hours, or from about 30 hours to about 60 hours, or from about 30 hours to about 50 hours.

[0046] In certain embodiments, the bispecific antibody-containing drug substance is gently mixed during thawing. Gentle mixing can be achieved, for example, by using a tilting shaker or by gently inverting the drug substance container. In certain embodiments, the bispecific antibody-containing drug substance 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 the 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, a bispecific antibody-containing drug substance has a pH ranging from about pH 3.5 to about pH 7.5 or from about pH 4.0 to about pH 7.0. In certain embodiments, a bispecific antibody-containing drug substance has a pH ranging from about pH 4.0 to about pH 6.5. In certain embodiments, a bispecific antibody-containing drug substance has a pH ranging from about pH 4.0 to about pH 4.8. In certain embodiments, a 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 filtration 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), in which 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 the bispecific antibody, 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 present methods 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, a 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 comprises: The method further comprises dividing the product into formulations. Such formulations may be presented in unit dosage forms, for example, in ampoules, single-dose containers, or multi-dose containers. The formulations may, if desired, be presented in a vial, pack, or dispenser device which may contain one or more unit dosage forms containing the bispecific antibody.

[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 dosage forms. 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, e.g., Niven, R., Prestrelski, S.J., Treuheit, M.J., Ip, A.Y. 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 occur before or after the division step.

[0054] The method disclosed herein reduces aggregates of bispecific antibodies. The method is based on the surprising finding that when thawed antibodies are 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), aggregates formed during storage under frozen conditions (e.g., about -20°C to about -40°C) are reduced. Without wishing to be bound by any theory, it is believed that the aggregates return to a non-aggregated state after the storage 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 bispecific antibody-containing drug substance 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 bispecific antibody-containing drug substance contains less than about 1% bispecific antibody dimers after the holding period. In certain embodiments, the bispecific antibody-containing drug substance contains less than about 0.5% bispecific antibody dimers after the holding period.

[0057] The methods disclosed herein do not affect or 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 as the bispecific antibody or 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 charge variant as the bispecific antibody or drug substance before freezing. In certain embodiments, the method produces a bispecific antibody or bispecific antibody-containing drug substance with the same or substantially the same potency as the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the method produces a bispecific antibody or bispecific antibody-containing drug substance with the same or substantially the same clipping level as the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the method produces a bispecific antibody or bispecific antibody-containing drug substance with the same or substantially the same chemical modifications (e.g., glycosylation) as the bispecific antibody or bispecific antibody-containing drug substance before freezing. In certain embodiments, the method produces a bispecific antibody or bispecific antibody-containing drug substance with the same or substantially the same pH as the bispecific antibody or bispecific antibody-containing drug substance before freezing.

[0058] bispecific antibody Bispecific antibodies that can 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, 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 terms "specifically binding domain" or "binding domain" are 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 enable 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 framework of the antibody VL and antibody VH. The term includes fragments of full-length antibodies and antibody variants. Examples of antibody fragments, antibody variants, or binding domains include: (1) Fab fragments, monovalent fragments containing VL, VH, CL, and CH1 domains; (2) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; (3) Fd fragments containing two VH and CH1 domains; (4) Fv fragments containing the VL and VH domains of a single antibody arm; (5) dAb fragments containing a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fvs (scFvs), the latter being preferred (e.g., from scFv libraries). Additional 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 derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. Thus, an antibody construct is 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, s Also included are cFv-zippers, 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 which specifically binds to an antigen or epitope independent of other V regions or domains.

[0061] As used herein, the terms "single-chain Fv," "single-chain antibody," or "scFv" are 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. Typically, single-chain antibodies further contain a polypeptide linker between the VH and VL domains that enables them to form the desired structure allowing for 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 for producing single chain antibodies are known, including those described in U.S. Pat. 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. 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 an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, as described herein, that allows them to be produced recombinantly as a single protein chain in which the VL and VH regions pair to form a monovalent molecule; see, e.g., 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 manner as intact or full-length antibodies. Thus, scFvs are typically fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains 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 connect 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 the 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, e.g., Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules using a linker peptide that is too short (e.g., about 5 amino acids) to allow the two variable regions to fold together, resulting in dimerization of the scFv. This type is known as a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences). 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 type of oligomer.

[0065] In certain embodiments, either the first, second, or first and second domains may comprise a single domain antibody, each comprising a variable domain or at least a CDR of a single domain antibody. Single domain antibodies comprise 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 derived VHH fragments from them. NARHeavy chain antibodies (IgNARs) have the ability to generate single domain antibodies, called fragments. An alternative approach is to split the dimeric variable domains from common immunoglobulins, for example from humans or rodents, into monomers, thereby obtaining VH or VL as single domain Abs. While most research on single domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to 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 may be presented on the cell surface with an extracellular portion, and often have both transmembrane and cytoplasmic portions of the molecule. These antigens may be presented only by tumor cells and never by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may exhibit 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, and they are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be 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: : (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 that binds to an extracellular epitope of the human CD3 epsilon chain and comprises 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) SEQ ID NO: 156 of WO 2008 / 119567 CDR-H1 as set forth in SEQ ID NO: 157 of WO 2008 / 119567, CDR-H2 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 (30) groups 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] Preferably, the second domain that 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 to 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 CD3-binding second domain 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 domains have the following format: a pair of VH and VL domains in the format of a single-chain antibody (scFv). The VH and VL domains are arranged in the order of VH-VL or VL-VH. Preferably, the VH domain is arranged at the N-terminus of the linker sequence and the VL domain is arranged at the C-terminus of 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 "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 retaining at least a functional portion of one CH2 domain and one functional portion of one CH3 domain, with 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, with the hinge region being amino-terminal to the CH2 domain. The hinge region of a bispecific antibody is thought to promote dimerization. Such Fc polypeptide molecules can 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 may be a polypeptide region comprising a portion of the CH2 region and the CH3 region. Such Fc polypeptide molecules may 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 is an amino acid sequence as disclosed in WO 2014 / 153063. The Fc monomer has the amino acid sequence: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, Two Fc moieties 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 the scFc domain.

[0079] IgG hinge regions can be identified by similarity using Kabat numbering as shown in Table 1. It is envisioned 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 Kabat numbering. It is also envisioned 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 of D234 to P243 as shown in Table 1 below - sequence variations are also envisioned, provided 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 eliminated by an N314X substitution, where X is any amino acid except Q. This substitution is preferably an 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). An IgG1 subtype hinge sequence may have the following sequence EPKSCDKTHTCPPCP (as shown in Table 1 and in SEQ ID NO: 196). Accordingly, 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 a first domain with a second domain, or to fuse a first or second domain with a third domain, the linker is preferably of sufficient length and sequence to ensure that each of the first and second domains retains its distinct binding specificity independently of the 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, e.g., 12 or fewer amino acid residues. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues are preferred. Peptide linkers of fewer than 5 amino acids are contemplated, containing 4, 3, 2, or 1 amino acid, with Gly-rich linkers being preferred.

[0086] A particularly preferred "single" amino acid "peptide linker" is Gly. Thus, the peptide linker may consist of the 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 fusing the first and second domains has the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 197). A preferred embodiment of the peptide linker for fusing the second and third domains is a (Gly)4-linker and a G4-linker, respectively.

[0088] The peptide linker by which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomer") are fused to one another 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, and 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 is an amino to carboxyl group. In order of sill: The HLE domain has the structure hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0090] In certain embodiments, 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 having the general structure RSSR. This linkage, also known as an S-S 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 that form 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 an intradomain cysteine ​​disulfide bridge 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 an 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 antibodies 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, preferably N314G, as described above, and / or the further substitutions V321C and R309C.

[0095] Bispecific antibodies may also comprise additional domains, for example, that aid in the isolation of the molecule or that are relevant to tailoring the pharmacokinetic profile of the molecule. Domains that aid in the isolation of the antibody construct may be selected from peptide motifs or secondarily introduced moieties that can be captured by isolation methods, for example, separation columns. Non-limiting examples of such additional domains include the 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., Strep II tags), and His tags. All of the bispecific antibodies disclosed herein are characterized by a sequence of consecutive His residues, preferably five, more preferably six His residues (hexa-His) in the amino acid sequence of the molecule. The His tag may comprise a His-tag domain, commonly known as a repeat of hexahistidine (HHHHHH). The His tag can be placed, for example, at either the N- or C-terminus of the antibody construct, but is preferably placed 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 may 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 of such binding domains comprising two antibody variable domains have been described above and include, for example, the Fv, scFv, or Fab fragments described hereinabove. Alternatively, either or both of the binding domains may comprise only a single variable domain. Examples of such single domain binding domains have been described hereinabove 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, wherein the first domain binds to CD70, the second domain binds to human CD3, and the third domain is an HLE domain having, in amino to carboxyl order: 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, wherein the first domain binds to a tumor antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, BCMA, or PSMA, the second domain binds to human CD3, and the third domain is an HLE domain having, in amino to carboxyl order: 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 by having an amino acid sequence selected from the following:

[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 extending (HLE) domain, preferably an scFc domain, a heterozygous Fc 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 to CD70 and a second domain that binds to CD3. In one embodiment, the first domain binds to CD70 and has CDRs as set forth in SEQ ID NOs: 182-187, and the second domain binds to 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 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 to BCMA and a second domain that binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In embodiments, the first domain binds to 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 to CD33 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 CD33 and has CDRs as set forth in SEQ ID NOs: 29-31 and 34-36, and the second domain binds to 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 to EGFRvIII 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 EGFRvIII and has CDRs as set forth in SEQ ID NOs: 42-47, and the second domain binds to 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 an 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, which comprises a first domain that binds to CDH19 and a second domain that binds to CD3. In one embodiment, 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 to FLT3 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 FLT3 and has CDRs as set forth in SEQ ID NOs: 83-88, and the second domain binds to 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 FLT3xCD3 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 to PSMA 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 PSMA and has CDRs as set forth in SEQ ID NOs: 137-142, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOs: 9-14. In one embodiment, the PSMAxCD3 bispecific antibody comprises a VH and 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 to 151, 161 to 168, and 176 to 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, by the methods disclosed in WO 2008 / 119657 and WO 2017 / 134140.

[0114] In some embodiments, the bispecific antibody is a masked bispecific antigen-binding protein. Masked bispecific antigen-binding proteins have been previously described. See, e.g., WO 2017 / 040344; U.S. Patent Application Publication No. 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 comprising a masking domain (MD) coupled (e.g., via 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) comprising 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 of the AB domain to the antigen is increased or induced. Masked antigen binding proteins have been previously described, for example, in WO 2017 / 040344, U.S. Patent No. 9,540,440, U.S. Patent Application Publication No. 20150118254, U.S. Patent No. 9,127,053, U.S. Patent No. 9,517,276, and U.S. Patent 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 because the PR is cleaved by a protease, thereby releasing at least the 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)).

[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 configuration 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 a naive state and in the presence of antigen, binding of the AB to the antigen is reduced or inhibited, 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 translocates 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 by, for example, 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 coexisting in tissue with the desired antigen of the masked antigen-binding protein AB (including, for example, tumor cells). In some embodiments, the protease is u-type plasminogen activator (uPA, also known as urokinase), legumain, and / or matriptase (also known as MT-SP1 or MTSP1). In some embodiments, the protease is a matrix metalloproteinase (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 a different 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 a different 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 the 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) comprising 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 bispecific masked antigen-binding proteins, 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 circulation and are activated at sites of therapeutic and / or diagnostic interest, but are not activated in normal (i.e. 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 antibody equivalent 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 cell. 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) < 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 comprising 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 (scFvs) are antigen-binding proteins in which the VL and VH regions are linked via a linker (e.g., a synthetic sequence of amino acid residues, usually about 15 to about 20 amino acids in length) to form a continuous protein chain, where the linker 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 exemplary linkers contain at least 4-5 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 suitable linker for linking the scFv to the heavy chain variable region is GGGGS (SEQ ID NO: 197). Typical linkers contain at least one suitable linker having four to five amino acids.

[0128] The masked antigen-binding protein may be attached (e.g., by covalent or other means) to the antigen-binding protein. The present invention includes a masking domain (MD) coupled to an antibody (AB). The masking domain (MD) comprises a masking peptide (or masking polypeptide) (MP) and a protein recognition site (PR). The masking peptide (or masking polypeptide) can be a stretch of amino acids that prevents the antigen-binding domain from binding to its antigen. Generally, masking peptides, short sequences of 5 to 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 Application 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 to EGFR); and U.S. Patent Application 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 to 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 connects 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, e.g., tumor cells) near cells expressing the target and / or produced by cells (including, e.g., 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 known as urokinase), legumain, and / or matriptase (also known as 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 undergoes a conformational change upon interaction with, for example, a protein or protease, such that the position of the MP is modulated and the AB is free to bind to the target.

[0130] In various aspects of the present disclosure, a masked bispecific antigen-binding protein comprises an 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 independently the same or different. For example, a masked bispecific antigen-binding protein may comprise (i) two scFvs that bind to CD3 and MD1 linked to each scFv (optionally the same MD for each scFv), and (ii) two Fab portions that bind to EGFR and MD2 linked to each Fab (optionally 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- or C-terminus of the antigen-binding domains, as long as the MDs are capable of interfering with 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 can 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 with 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 and U.S. Patent Application Publication No. 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, 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 an intact antibody structure, 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 linked to an MD; if both scFvs are linked to an MD, the MDs can be the same or different (i.e., in various aspects, the MP and / or PR are different, but the PRs are preferably 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. [Example]

[0169] Example 1: Increased aggregation of BiTE molecules when stored under frozen conditions (-20°C) Compositions containing 1 mg / mL each of HLE BiTEs (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 their hydrodynamic volume using a size-exclusion ultra-high performance analytical column. High molecular weight (aggregate peak) elutes earlier than monomer and lower molecular weight peaks. Components eluted isocratically, detected by UV detection, and integrated. 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 at -20°C for 1 month. After 1 month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates; the results 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 level of HMW aggregates. The results are shown in Figure 2B. When BiTE was stored at -10°C, -20°C, or -30°C, the level of HMW aggregates increased. A slight increase in HMW aggregate levels was observed when BiTE was stored at temperatures lower than the Tg' of the BiTE-containing composition (-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 HMW aggregate levels. 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 storage period, HMW aggregate levels had decreased to less than 5% (Figure 3A).

[0173] In a second series 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 level 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 decreased to less than 5% after the storage period (Figure 3B).

[0174] In a third series of experiments, HLE BiTEs (MSLNxCD3, CD19xCD3, CD33xCD3, CDH19xCD3, BCMAxCD3, D) were added at a concentration of 1 mg / mL. Compositions containing each of the CD31-dependent markers (LL3xCD3, FLT3xCD3, PSMAxCD3, and CD70xCD3) 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. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. The level of HMW aggregates in each composition decreased to less than 1% after the storage period (Figure 3C).

[0175] In a fourth series of experiments, compositions containing each of the HLE BiTEs (CD33xCD3 and DLL3xCD3) at a concentration of 1 mg / mL were filled into 5 mL volumes 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 level 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 level of HMW aggregates in each composition decreased 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 sample was determined by comparing the response of the test sample to that of a reference standard (relative potency).

[0178] Reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) was used to measure clipping of BiTE molecules. 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 SDS-coated proteins were separated based on their hydrodynamic size; smaller proteins migrated faster than larger proteins. A photodiode array (PDA) detector was used to detect and integrate proteins, and results are reported as the relative peak area percentages of the low molecular weight, main peak, and high molecular weight peak.

[0179] Charge variants of BiTE molecules were measured using cation exchange high-performance liquid chromatography (CEX-HPLC). Protein charge variants were eluted using a mobile phase gradient of increasing ionic strength at the appropriate pH. Proteins with less positive surface charge eluted 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 a percentage of the total peak area.

[0180] As shown in Table 4, storage of thawed BiTE did not negatively affect stability, while 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 1 month at -20° C. After 1 month, the composition was thawed at room temperature and immediately analyzed by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) to determine the level of HMW aggregates (e.g., bispecific antibody dimers) in the composition.

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

[0184] Example 6: Stabilizing effect of benzyl alcohol (BA) Compositions containing 1 mg / mL 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 the level of HMW aggregates. The presence of BAs stabilizes BiTEs during storage (Figure 5).

[0185] All references cited in this application are incorporated herein by reference. The present invention includes, but is not limited to, the following aspects. [Aspect 1] 1. A method for reducing aggregates of a bispecific antibody, comprising: 1. A method comprising maintaining a 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 claim 1, wherein the bispecific antibody is maintained at the temperature for 4 hours to 96 hours. [Aspect 4] Aspect 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] 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] Aspect 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] Aspect 8. The method of any one of aspects 1 to 7, wherein the aggregates comprise high molecular weight (HMW) aggregates. [Aspect 9] Aspect 9. The method of any one of aspects 1 to 8, wherein said bispecific antibody comprises less than about 1% of said HMW aggregates after said holding period. [Aspect 10] 10. The method of embodiment 9, wherein said 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: Thawing the bispecific antibody-containing drug substance stored at a temperature of -20°C to -40°C maintaining the thawed drug substance containing the bispecific antibody at a temperature of 5°C to 45°C for at least 4 hours; A method comprising: [Aspect 13] 13. The method of aspect 12, wherein the composition is a pharmaceutical composition comprising the bispecific antibody. [Aspect 14] 14. The method of aspect 12 or 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 of any one of aspects 12 to 15, wherein the drug substance is maintained at said temperature for a period of 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 of 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 of any one of aspects 12 to 18, wherein the drug substance has been stored at a temperature of from -20°C to -35°C. [Aspect 20] 20. The method of any one of aspects 12 to 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 holding a thawed drug substance comprising the 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 aspects 21 to 22, wherein the drug substance has been frozen at about −32° C. [Aspect 24] 24. The method of any one of aspects 21 to 23, wherein the drug substance is maintained at said temperature for a period of 4 hours to 96 hours. [Aspect 25] 25. The method of 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 of any one of aspects 21 to 25, wherein the drug substance is maintained at the same temperature as the temperature at which the drug substance is thawed. [Aspect 27] 27. The method of aspect 26, wherein the drug substance is thawed and maintained at a temperature of 15°C to 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 aspect 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 the HMW aggregate comprises a dimer of the bispecific antibody. [Aspect 32] 32. The method according to any one of aspects 1 to 31, wherein the drug 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] A method according to any one of aspects 1 to 34, wherein the bispecific antibody is 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 aspect 35, wherein the target cell surface antigen is CD19, CD33, or BCMA. [Aspect 37] 37. The method of claim 36, wherein the first binding domain comprises a VH region and a VL region; 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] Aspect 39. The method of 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 wherein 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] 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 claim 42, wherein the first binding domain comprises a VH region and a VL region; 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. whether it contains columns; 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] Aspect 35. 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. A method according to 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, wherein the MD1 is (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 is (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: [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 the AB1 binds to human CD3 and the AB2 binds to human EGFR.

Claims

[Claim 1] An invention described in the specification or drawings.

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