Anti-FGFR2 antibody formulation

Abstract

The present disclosure relates to a formulation of an anti-fibroblast growth factor receptor 2 (FGFR2) antibody that can be stored as a liquid in some embodiments, for example, in a form ready for immediate use, and can be administered intravenously in some embodiments, for example, by intravenous (IV) infusion.

Description

Technology Field

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 741,772 filed October 5, 2018, the full text of which is by reference.

[0002] Technology field

[0003] The present disclosure relates to a formulation for an anti-FGFR2 (fibroblast growth factor receptor 2) antibody, e.g., an anti-FGFR2-IIIb antibody, which, in some embodiments, can be stored for a long period in a form that is immediately available for use, e.g., as a liquid, and in some embodiments can be administered intravenously, e.g., by intravenous (IV) infusion. Background Technology

[0004] The present disclosure relates to specific formulations of antibodies that bind to FGFR2, e.g., the spliced ​​form FGFR2-IIIb (fibroblast growth factor receptor 2 IIIb). FGFR2 is one of four FGFR proteins (FGFR1, 2, 3, and 4). FGFR is characterized by multiple alternative splicing of its mRNA, resulting in various isoforms (Ornitz et al., J. Biol. Chem. 271:15292, 1996; see also Swiss-Prot P21802 and isoforms P21802-1 to P21802-20 for the sequences of FGFR2 and its isoforms). In the case of FGFR2, selective splicing results in, for example, the isoforms FGFR2-IIIb and FGFR2-IIIc (or directly FGFR2b and FGFR2c). The FGFR2-IIIb form of FGFR2 (or denoted as K-sam-II) is a high-affinity receptor for both FGF1 and KGF family members (FGF7, FGF10, and FGF22), whereas FGFR2-IIIc (also denoted as K-sam-I) binds well to both FGF1 and FGF2 but does not bind to KGF family members (Miki et al., Proc. Natl. Acad. Sci. USA 89:246, 1992). FGFR2-IIIb is the only receptor for KGF family members (Ornitz et al., 1996, op. cit Therefore, it is also designated as KGFR.

[0005] Inhibitors of FGFR2 may include antibodies. For example, U.S. Patent No. 8,101,723 B2 describes, for example, monoclonal antibodies that bind to human FGFR2-IIIb but bind less well or not to FGFR2-IIIc, and vice versa. U.S. Patent Publication No. 2015-0050273 A1 describes specific afucosylated antibodies that bind to FGFR2-IIIb. The present disclosure describes formulations that may be used for antibodies described, for example, in U.S. Patent No. 8,101,723 B2 and U.S. Patent Publication No. 2015-0050273 A1.

[0006] In some embodiments, formulation conditions have been identified in this specification such that the anti-FGFR2 antibody described herein is prepared in a liquid formulation, remains stable after storage for several months, and can be administered intravenously, e.g., by IV infusion.

[0007] The present disclosure comprises, for example, a pharmaceutical formulation of an anti-FGFR2 antibody comprising: i) 10 to 30 mg / ml antibody; ii) 5 to 40 mM of a buffer selected from one or more of histidine, citrate, or phosphate; iii) 130 to 170 mM arginine or 250 to 290 mM sucrose; and iv) 0.002% to 0.1% polysorbate 20 or polysorbate 80; wherein the formulation has a pH of 5.0 to 6.5, and the anti-FGFR2 antibody comprises: a) an antibody comprising a heavy chain having the sequence of SEQ ID NO. 2 and a light chain having the sequence of SEQ ID NO. 3; b) an antibody comprising a heavy chain (HC) supervariable region 1 (HVR1) comprising the sequence of SEQ ID NO. 6, an HC HVR2 comprising the sequence of SEQ ID NO. 7, and an HC HVR3 comprising the sequence of SEQ ID NO. 8, and a light chain (LC) comprising HVR1 comprising the sequence of SEQ ID NO. 9, an LC HVR2 comprising the sequence of SEQ ID NO. 10, and an LC HVR3 comprising the sequence of SEQ ID NO. 11; and c) an antibody comprising a heavy chain comprising a variable region sequence comprising the sequence of SEQ ID NO. 4 and a light chain comprising a light chain variable region sequence comprising the sequence of SEQ ID NO. 5. In some embodiments, the formulation has one or more of the following characteristics: (a) is a liquid formulation ready for immediate use; (b) is not lyophilized before administration to a patient; (c) is contained in a single-use vial; (d) protein aggregation in the formulation increases by 2.0% or less after storage at 5°C for 6 months; (e) Increase in protein aggregation in the formulation by 2.0% or 2.5% or less after storage at 25°C for 3 months; (f) Increase in protein aggregation in the formulation by 7.0% or less after storage at 40°C for 3 months; (g) No change in charged protein variants in the formulation by more than 5% after storage at 5°C for 6 months; (h) Protein aggregation in the formulation by 2.0% after 5 freeze-thaw cycles at -70°C.Increase of 0% or less; (i) Increase in protein aggregation in the formulation of 2.0% or less after 72 hours of mechanical stress at 500 rpm; (j) Diluted with saline solution before intravenous administration; (k) Administered intravenously (e.g., by intravenous infusion); (l) Isotonic with human plasma.

[0008] In some cases, the formulation comprises 10 to 15 mg / ml, 15 to 20 mg / ml, 20 to 25 mg / ml, 18 to 22 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml of anti-FGFR2 antibody. In some cases, the formulation comprises 10 to 40 mM, 10 to 30 mM, 15 to 25 mM, 10 to 20 mM, 20 to 30 mM, 18 to 22 mM, 10 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM of buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the formulation comprises 130 to 150 mM, 150 to 170 mM, 140 to 160 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, or 170 mM arginine. In some cases, the formulation comprising arginine does not comprise sucrose. In some embodiments, the pH of the arginine-containing formulation is 5.0 to 7.0, 5.0 to 6.0, 5.5 to 6.0, 5.5 to 6.5, 5.5 to 5.9, 5.6 to 5.8, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some cases, the pH of the formulation is 5.5 to 5.9, 5.6 to 5.8, 5.6, 5.7, 5.8, 5.9, or 6.0. In some embodiments, the formulation comprises 260 to 280 mM, 250 mM, 260 mM, 270 mM, 280 mM, and 290 mM of sucrose. In some such cases, the formulation comprising sucrose does not contain arginine.In some embodiments, the pH of the sucrose-containing formulation is 5.0 to 7.0, 5.0 to 6.0, 5.5 to 6.0, 5.5 to 5.9, 5.6 to 5.8, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH of the formulation is 5.8 to 6.2, 5.9 to 6.1, 5.9, 6.0, or 6.1.

[0009] Any of the above formulations may also comprise 0.01 to 0.1%, 0.005 to 0.05%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 20 or 80. In some cases, the formulation comprises 0.01 to 0.1%, 0.005 to 0.05%, 0.002%, 0.003%. It contains 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 20.

[0010] In some embodiments, the formulation essentially consists of an anti-FGFR2 antibody; a citrate, phosphate, or histidine buffer; arginine or sucrose; and polysorbate 20 or 80. In some such cases, the formulation essentially consists of an anti-FGFR2 antibody, histidine, arginine or sucrose, and polysorbate 20.

[0011] The present disclosure also comprises, for example, a pharmaceutical formulation of an anti-FGFR2 antibody, wherein the formulation is a liquid formulation not lyophilized before use, comprising i) 10 to 25 mg / mL of anti-FGFR2 antibody; ii) 10 to 30 mM histidine buffer; iii) 140 to 160 mM arginine; and iv) 0.005% to 0.05% polysorbate 20; and the formulation has a pH of 5.6 to 5.8. The present disclosure also comprises, for example, a pharmaceutical formulation of an anti-FGFR2 antibody, wherein the formulation comprises i) 10 to 25 mg / mL of anti-FGFR2 antibody; ii) 20 mM histidine buffer; iii) 150 mM arginine; and iv) a liquid formulation that is not lyophilized before use, comprising 0.01% polysorbate 20; the formulation has a pH of 5.7. Embodiments of this specification further comprise a pharmaceutical formulation of an anti-fibroblast growth factor receptor 2 (FGFR2) antibody comprising i) 20 mg / ml anti-FGFR2 antibody; ii) 20 mM L-histidine; iii) 150 mM L-arginine; and iv) 0.01% polysorbate 20, wherein the pharmaceutical formulation is a liquid formulation that is not lyophilized before use and has a pH of 5.7. In any of these cases, in some embodiments, the formulation essentially consists of the anti-FGFR2 antibody, histidine, arginine, and polysorbate 20.In some such embodiments, the anti-FGFR2 antibody may be selected from a) an antibody comprising a heavy chain having the sequence of SEQ ID NO. 2 and a light chain having the sequence of SEQ ID NO. 3; b) an antibody comprising a heavy chain (HC) having a supervariable region 1 (HVR1) having the sequence of SEQ ID NO. 6, an HC HVR2 having the sequence of SEQ ID NO. 7, and an HC HVR3 having the sequence of SEQ ID NO. 8, and a light chain (LC) having a light chain having a sequence of SEQ ID NO. 9, an LC HVR2 having the sequence of SEQ ID NO. 10, and an LC HVR3 having the sequence of SEQ ID NO. 11; and c) an antibody comprising a heavy chain having a variable region sequence having the sequence of SEQ ID NO. 4 and a light chain having a light chain variable region sequence having the sequence of SEQ ID NO. 5. The pharmaceutical formulation described above may have one or more of the following characteristics: (a) contained in a single-use vial; (b) increase in protein aggregation in the formulation by 2.0% or less after storage at 5°C for 6 months; (c) increase in protein aggregation in the formulation by 2.0% or 2.5% or less after storage at 25°C for 3 months; (d) increase in protein aggregation in the formulation by 7.0% or less after storage at 40°C for 3 months; (e) no change in charged protein variants in the formulation by more than 5% after storage at 5°C for 6 months; (f) increase in protein aggregation in the formulation by 2.0% or less after 5 freeze-thaw cycles at -70°C; (g) increase in protein aggregation in the formulation by 2.0% or less after 72 hours of mechanical stress at 500 rpm; (h) diluted with saline solution before intravenous administration; (i) administered intravenously (e.g., by intravenous infusion); and (j) isotonic with human plasma.The pharmaceutical formulation comprising 10 to 25 mg / ml of antibody may, more specifically, comprise 10 to 15 mg / ml, 15 to 20 mg / ml, 20 to 25 mg / ml, 18 to 22 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml of anti-FGFR2 antibody.

[0012] The present disclosure also comprises, for example, a pharmaceutical formulation of an anti-FGFR2 antibody, wherein the formulation is a liquid formulation not lyophilized before use, comprising i) 10 to 25 mg / mL of anti-FGFR2 antibody; ii) 10 to 30 mM histidine buffer; iii) 260 to 280 mM sucrose; and iv) 0.005% to 0.05% polysorbate 20; and the formulation has a pH of 5.8 to 6.2. The present disclosure also comprises, for example, a pharmaceutical formulation of an anti-FGFR2 antibody, wherein the formulation comprises i) 10 to 25 mg / mL of anti-FGFR2 antibody; ii) 20 mM histidine buffer; iii) 270 mM sucrose; and iv) a liquid formulation that is not lyophilized before use, comprising 0.01% polysorbate 20; and the formulation has a pH of 6.0. In further embodiments, the pharmaceutical formulation may comprise i) 20 mg / ml anti-FGFR2 antibody; ii) 20 mM L-histidine; iii) 270 mM sucrose; and iv) 0.01% polysorbate 20, wherein the formulation is a liquid formulation that is not lyophilized before use and has a pH of 6.0. In some such embodiments, the formulation essentially consists of the anti-FGFR2 antibody, histidine, sucrose, and polysorbate 20.In some such embodiments, the anti-FGFR2 antibody may be selected from a) an antibody comprising a heavy chain having the sequence of SEQ ID NO. 2 and a light chain having the sequence of SEQ ID NO. 3; b) an antibody comprising a heavy chain (HC) having a supervariable region 1 (HVR1) having the sequence of SEQ ID NO. 6, an HC HVR2 having the sequence of SEQ ID NO. 7, and an HC HVR3 having the sequence of SEQ ID NO. 8, and a light chain (LC) having a light chain having a sequence of SEQ ID NO. 9, an LC HVR2 having the sequence of SEQ ID NO. 10, and an LC HVR3 having the sequence of SEQ ID NO. 11; and c) an antibody comprising a heavy chain having a variable region sequence having the sequence of SEQ ID NO. 4 and a light chain having a light chain variable region sequence having the sequence of SEQ ID NO. 5. The pharmaceutical formulation described above may have one or more of the following characteristics: (a) contained in a single-use vial; (b) increase in protein aggregation in the formulation by 2.0% or less after storage at 5°C for 6 months; (c) increase in protein aggregation in the formulation by 2.0% or 2.5% or less after storage at 25°C for 3 months; (d) increase in protein aggregation in the formulation by 7.0% or less after storage at 40°C for 3 months; (e) no change in charged protein variants in the formulation by more than 5% after storage at 5°C for 6 months; (f) increase in protein aggregation in the formulation by 2.0% or less after 5 freeze-thaw cycles at -70°C; (g) increase in protein aggregation in the formulation by 2.0% or less after 72 hours of mechanical stress at 500 rpm; (h) diluted with saline solution before intravenous administration; (i) administered intravenously (e.g., by intravenous infusion); and (j) isotonic with human plasma.The pharmaceutical formulation comprising 10 to 25 mg / ml of antibody may, more specifically, comprise 10 to 15 mg / ml, 15 to 20 mg / ml, 20 to 25 mg / ml, 18 to 22 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, or 25 mg / ml of anti-FGFR2 antibody.

[0013] In some embodiments of the present disclosure, the pharmaceutical formulation may not include one or more of the following: a sugar other than sucrose, a sugar alcohol, a protein species other than an anti-FGFR2 antibody, a surfactant other than polysorbate 20 or polysorbate 80, an amino acid other than arginine and histidine, Cu 2 + , Mg 2 + , and Mn 2 + In any embodiment of this disclosure, the formulation may be contained in a single-use vial.

[0014] The present disclosure also includes a method for treating a solid tumor in a patient in need, comprising the step of administering an effective amount of the pharmaceutical formulation of the present specification to the patient. In some embodiments, the formulation is administered to the patient intravenously (e.g., by intravenous infusion).

[0015] In any formulation of this specification, the anti-FGFR2 antibody may be optionally defucosylated. In any formulation of this specification, the anti-FGFR2 antibody may be a full-length antibody or an antigen-binding fragment, e.g., Fv, single-chain Fv(scFv), Fab, Fab', or (Fab')2. In any formulation of this specification, the antibody may be a chimeric, humanized, or human antibody. In any formulation of this specification, the antibody may be bispecific, multispecific, or conjugated. Brief explanation of the drawing

[0016] Fig. 1 The mean binding of defucosylated anti-FGFR2IIIb antibodies to FGFR2IIIb or protein A by Biacore® assay after incubation of antibodies at different concentrations of hydrogen peroxide (0.01%, 0.1%, and 1.0%, as well as 0% (control)). Incubation of anti-FGFR2IIIb antibodies with hydrogen peroxide interfered with protein A binding to some extent, but did not interfere with binding to FGFR2IIIb. Fig. 2 Differential scanning calorimetry (DSC) thermograms of the citrate / NaCl formulation of anti-FGFR2IIIb antibody at pH 4.0, 5.0, and 6.0. The data indicate that the unfolding temperature of the formulation increases with pH in this range. Fig. 3 Size exclusion high-performance liquid chromatography (SE-HPLC) chromatograms of anti-FGFR2IIIb antibody screening samples at pH 4, 5, 6, 7, and 8 as discussed in Example 5 below. Fig. 4 The effect of buffer pH (in citrate or phosphate NaCl buffer) on aggregate formation determined by SE-HPLC in the range of pH 4 to 8 and over a storage period of 0 to 2 months at 40°C. The figure shows a large % increase in aggregates at pH 4 compared to other pH values. Fig. 5The effect of buffer pH (in citrate or phosphate NaCl buffer) on clip (fragment) formation determined by SE-HPLC in the range of pH 4 to 8 and over a storage period of 0 to 2 months at 40°C. The figure above shows a large % increase in clip formation at pH 4 compared to other pH values. Fig. 6 The effect of buffer pH (in citrate or phosphate NaCl buffer) on aggregate formation determined by SE-HPLC in the range of pH 4 to 8 and over a storage period of 0 to 2 months at 25°C. The figure indicates that, with the exception of the pH 8 formulation, where aggregates increased from about 2.5% to about 4% over 2 months of storage, all formulations generally contained less than 3.0% aggregates over this period. Fig. 7 The effect of buffer pH (in citrate or phosphate NaCl buffer) on clip (fragment) formation determined by SE-HPLC over a storage period of 0 to 2 months in the range of pH 4 to 8 and at 25°C. The figure shows that at pH 4, the clip increased to nearly 1.5% compared to other pH values ​​that maintained less than 0.5% clip. Fig. 8 Representative weak cation exchange (WCX)-HPLC chromatogram of the anti-FGFR2IIIb formulation for evaluating the charged variant profile. Fig. 9 The effect of buffer pH (in citrate or phosphate NaCl buffer) on acid variant formation determined by WCX-HPLC in the range of pH 4 to 8 and over a storage period of 0 to 2 months at 25°C. There was a relatively large % increase in acid variants in the pH 8 formulation, and the pH 7 formulation showed the next largest % increase. Fig. 10The effect of buffer pH (in citrate or phosphate NaCl buffer) on the formation of basic variants determined by WCX-HPLC in the pH range of 4 to 8 and over a storage period of 0 to 2 months at 25°C. There was a relatively large % decrease in basic variants in the pH 8 formulation, and the pH 7 formulation showed the next largest % decrease. The smallest % change was observed at pH 4. Fig. 11 The effect of buffer pH (in citrate or phosphate NaCl buffer) on the size of the main peak of the WCX-HPLC chromatogram in the range of pH 4 to 8 and over a storage period of 0 to 2 months at 25°C. The largest % change in the main peak was observed in the pH 4 and pH 8 formulations, and the smallest % change was observed in the pH 5, pH 6, and pH 7 formulations. Fig. 12 The appearance of the solutions of the seven anti-FGFR2IIIb formulations shown in Table 8 below after shaking at 500 rpm for 72 hours. Fig. 13 Effect of excipients on anti-FGFR2IIIb stability for 5 freeze-thaw cycles at 70°C to ambient temperature. The formulations are as provided in Table 8. Only the histidine / NaCl formulation (No. 4 in Table 8) appeared to show an increase in aggregates during multiple freeze-thaw cycles. Fig. 14 The effect of buffers and extenders on anti-FGFR2IIIb aggregate formulations over 1 month at 40°C as determined by SE-HPLC. Fig. 15 The effect of buffers and extenders on anti-FGFR2IIIb aggregate formulations over 3 months at 40°C as determined by SE-HPLC. Fig. 16 Effects of buffers and extenders on anti-FGFR2IIIb acidic variant formulations over 3 months at 25°C as determined by WCX-HPLC. Fig. 17 The effect of buffers and extenders on the size of major WCX-HPLC peaks over 3 months at 25°C. Fig. 18The effect of buffers and extenders on the size of major WCX-HPLC peaks over 3 months at 25°C. Fig. 19 The effect of mechanical stress over 72 hours on histidine / arginine or histidine / sucrose anti-FGFR2IIIb antibody formulations at pH 5.5 to 6.5 as determined by analysis of aggregates by SE-HPLC. Fig. 20 The effect of mechanical stress over 72 hours on histidine / arginine or histidine / sucrose anti-FGFR2IIIb antibody formulations at pH 5.5 to 6.5 as determined by analysis of clips (fragments) by SE-HPLC. Fig. 21 The effect of 5 freeze-thaw cycles at -70°C to ambient temperature on histidine / arginine or histidine / sucrose anti-FGFR2IIIb antibody formulations at pH 5.5 to 6.5 as determined by analysis of aggregates by SE-HPLC. Fig. 22 The effect of 5 freeze-thaw cycles at -70°C to ambient temperature on histidine / arginine or histidine / sucrose anti-FGFR2IIIb antibody formulations at pH 5.5 to 6.5 as determined by analysis of clips (fragments) by SE-HPLC. FIGS. 23A to 23C The effect of a pH between 5.5 and 6.5 on aggregate formation in a histidine / arginine formulation comprising 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. Fig. 23A It indicates aggregate formation, which indicates that the aggregate % was generally maintained at less than 3% after 1 month at 40°C at pH 5.5 to 6.0. Fig. 23B It indicates aggregate formation, which indicates that the aggregate % was generally maintained at less than 2.5% after 3 months at 25°C at pH 5.5 to 6.0. Fig. 23C It indicates aggregate formation, which means that after 6 months at 5°C at all pH, the aggregate % generally remained at about 1.5% and less than 2%. FIGS. 24A to 24CThe effect of a pH between 5.5 and 6.5 on a clip (fragment) in a histidine / arginine formulation comprising 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. Fig. 24A This indicates clip (fragment) formation, which means that the clip % was generally maintained at about 1% or less than 1% at pH 6 after 1 month at 40°C at all pH levels. Fig. 24B It indicates clip formation, which means that the clip % generally remained below 1.5% after 3 months at 25°C at all pH. Fig. 24C It indicates clip formation, which means that the clip % was generally less than 1% after 6 months at 5°C at all pH, and the clip % of the formulation was generally maintained at less than 0.5% at pH 5.7 or 6.0. FIGS. 25A to 25C The effect of a pH between 5.5 and 6.5 on aggregate formation in a histidine / sucrose formulation comprising 20 mM histidine, 270 mM sucrose, and 0.01% polysorbate 20. Fig. 25A It indicates aggregate formation, which indicates that after one month at 40°C at pH 5.5 to 6.0, the aggregate % is generally maintained at less than 4%, less than 3% at pH 6.0 to 6.3, and about 2.5% at pH 5.5 and 5.7. Fig. 25B It indicates aggregate formation, which indicates that the aggregate % generally remained below 2.0% after 3 months at 25°C at pH 5.5 to 5.7 and the aggregate % remained below 2.5% at pH 6.0. Fig. 25C It indicates aggregate formation, which means that after 6 months at 5°C at all pH, the aggregate % generally remained at about 1.5% and less than 2%. FIGS. 26A to 26C The effect of a pH between 5.5 and 6.5 on a clip (fragment) in a histidine / sucrose formulation comprising 20 mM histidine, 270 mM sucrose, and 0.01% polysorbate 20. Fig. 26A This indicates clip (fragment) formation, which means that the clip % was generally maintained at less than 2% after 1 month at 40°C at all pH, or at less than 1.5% at pH 5.5 to 6.3. Fig. 26B This indicates clip formation, which indicates that clip % generally remained below 3.5% after 3 months at 25°C for all pH, but clip formation is pH-dependent, with the lowest percentage at 1, 2, and 3 months for the pH 5.5 formulation and the highest percentage at 3 months for the pH 6.3 formulation. Fig. 26C It indicates clip formation, which means that the clip % generally remained less than 1% after 6 months at 5°C at pH 6.0 or lower. FIGS. 27A to 27C The effect of a pH between 5.5 and 6.5 on the formation of acidic variants in a histidine / sucrose formulation comprising 20 mM histidine, 270 mM sucrose, and 0.01% polysorbate 20. Fig. 27A It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 40% after one month at 40°C at pH 5.5 to 6.0. Fig. 27B It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 30.0% after 3 months at 25°C at pH 5.5 to 6.0. Fig. 27C It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 25% after 6 months at 5°C at all pH levels. FIGS. 28A to 28C The effect of a pH between 5.5 and 6.5 on the formation of acidic variants in a histidine / arginine formulation comprising 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. Fig. 28A It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 40% after one month at 40°C at pH 5.5 to 6.0. Fig. 28B It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 30.0% after 3 months at 25°C at pH 5.5 to 6.0. Fig. 28C It indicates the formation of acidic variants, which means that the % of acidic variants generally remained below 25% after 6 months at 5°C at all pH levels. FIGS. 29A to 29C The effect of a pH between 5.5 and 6.5 on the formation of basic variants in a histidine / sucrose formulation comprising 20 mM histidine, 270 mM sucrose, and 0.01% polysorbate 20. Fig. 29A This indicates the formation of basic variants, which means that the % of basic variants remained mostly between 10% and 20% after one month at 40°C at pH 5.5 to 6.0. Fig. 29B This indicates the formation of basic variants, which means that the % of basic variants was generally maintained at 20% to 30% after 3 months at 25°C at pH 5.5 to 6.0. Fig. 29C It indicates the formation of basic variants, which means that the % of basic variants was generally maintained at 25% to 30% after 6 months at 5°C at all pH. FIGS. 30A to 30C The effect of a pH between 5.5 and 6.5 on the formation of basic variants in a histidine / arginine formulation comprising 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. Fig. 30A This indicates the formation of basic variants, which means that the % of basic variants remained mostly between 10% and 20% after one month at 40°C at pH 5.5 to 6.0. Fig. 30B This indicates the formation of basic variants, which means that the % of basic variants was generally maintained at 15% to 25% after 3 months at 25°C at pH 5.5 to 6.0. Fig. 30CIt indicates the formation of basic variants, which means that the % of basic variants was generally maintained at 25% to 30% after 6 months at 5°C at all pH. For further details regarding the above drawings, refer to the Examples section of this specification. Specific details for implementing the invention

[0017] Section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety for any purpose.

[0018] definition

[0019] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art. Additionally, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0020] In this application, the use of "or" means "and / or" unless otherwise noted. In the context of multiple dependent claims, the use of "or" merely refers to one or more prior independent or dependent claims as alternatives. Additionally, terms such as "element" or "component" include both elements and components comprising a single unit and elements and components comprising one or more subunits, unless otherwise specifically noted.

[0021] As described in this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the mentioned range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise indicated.

[0022] Units, prefixes, and symbols are indicated in the International System of Units (SI) accepted format. Numeric ranges include the digits that define the range. Measured values ​​are understood as approximations, taking into account the significant figures and error associated with the measurement.

[0023] The headings provided in this specification are not a limitation to the various aspects of the disclosure that may be included by referring to the entire specification. Accordingly, terms defined immediately below are more fully defined by referring to the specification in its entirety.

[0024] As used in accordance with the present disclosure, the following terms will be understood to have the following meanings unless otherwise indicated:

[0025] " administration "..." refers to the physical introduction of a composition containing a therapeutic agent into a subject using any various method and delivery system known to those skilled in the art. Common routes of administration for antibodies include, for example, by injection or infusion, intravenous (IV), intramuscular, subcutaneous (SC), intraperitoneal, spinal, or other parenteral administration routes. As used herein, the term "parenteral administration" generally refers to a mode of administration other than intestinal and local administration by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intravertebral, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtubercular, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intramedullary, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Non-parenteral routes include, for example, oral, intranasal, vaginal, rectal, sublingual, or local, local, epithelial, or mucosal administration routes. Administration also includes, for example, a single dose, multiple doses, and / or it may be performed over one or more extended periods.

[0026] As used herein, "pharmaceutical formulation" or " Therapeutic formulation"Speaks as follows: a composition comprising a therapeutic agent, such as an antibody, suitable for pharmaceutical use, such as being administered to a patient after being reconstituted, diluted, mixed, or dissolved, or thawed from a frozen state, either directly or with at least one additional composition. As used herein, " Available for immediate use one Since the formulation can be administered directly to the patient, it does not require dilution, reconstitution, thawing from a frozen state, dissolution in a solution, or mixing with other ingredients prior to administration.

[0027] As used in this specification, " subcutaneous administration " refers to the administration of a therapeutic agent under the skin. In some cases, subcutaneous administration (abbreviated as SC or SubQ) may be performed using a needle connected to a syringe or pump, or using a special type of infusion device (e.g., EpiPen®). In some cases, SC administration may release the therapeutic agent into the tissue between the skin and the muscle layer, and in some cases into the muscle layer.

[0028] As used in this specification, " Injectable one The formulation is suitable for administration by subcutaneous injection.

[0029] As used in this specification, " intravenously administration " refers to the direct administration of a therapeutic agent into the subject's vein. Intravenous administration is " intravenously injection It may be due to. Infusion refers to the use of pressure supplied by gravity to deliver a therapeutic agent to a patient.

[0030] The formulations of this specification are " vial"may be included in. In this specification, "vial" refers to any small container suitable for holding a liquid pharmaceutical formulation. In some cases, the "vial" may be connected to a syringe or pump, or the "vial" may be part of a delivery device, for example, for injecting the contents of the vial into a patient. In some embodiments, the formulation is " disposable vial "May be included in. "Single-use vial" refers to a "vial" in which the entire vial or the remaining contents of the vial are discarded after the contents are administered.

[0031] The term "when applied to a formulation, substance, or composition" freeze-dried " refers to a freeze-dried formulation, material, or composition.

[0032] As used in this specification, the term " Liquid formulation "It refers to a formulation in a liquid state, including, for example, a solution or a liquid emulsion.

[0033] As used in this specification, " buffer " is a substance that resists significant changes in pH upon dilution or the addition of acidic or basic components when added to a solution or formulation. Exemplary buffers in this specification include histidine, citrate, and phosphate buffers.

[0034] "as mentioned in this specification isotonicity The formulation is a formulation that has an osmotic pressure nearly equivalent to that of human body fluids present in the area where the formulation is introduced or administered, such as human plasma or human lymph.

[0035] As used in this specification, in the formulation " aggregation " or " Protein aggregation" refers to the aggregation of polypeptide molecules in a formulation. The amount of polypeptide in a formulation in an aggregated state, e.g., as a dimer or a multimer, may be expressed, e.g., as a percentage of the total polypeptide content of the formulation. Aggregation may be detected, e.g., by size exclusion chromatography or other techniques for separating proteins in solution based on size or molecular weight. In such techniques, " aggregates The amount of " may be equivalent to the amount of polypeptide in the formulation having a molecular weight at least twice that of the major polypeptide species of the formulation (e.g., anti-FGFR2-IIIb antibody). The species having a molecular weight twice that of the major polypeptide species of the formulation is referred to herein as " dimer While it can be considered as, a detectable species having a molecular weight greater than that of the dimer is " high molecular weight aggregates " or " Polymer " or may be referred to by similar terms.

[0036] In this specification, when referring to the storage of a formulation, the phrase " In the darkness "This means that the formulation is stored in a way that prevents exposure to ambient light and ultraviolet rays. For example, the formulation may be stored in a dark room or a dark space, or in packaging that protects the formulation against such light, or in a vial with walls made of or covered with a material that protects the contents against such light."

[0037] In some embodiments of this specification, a pharmaceutical formulation comprises one or more types of excipients or components. does not include In this context, the expression "does not include" means that the excluded component is not present above a trace level, for example, due to contaminants or impurities found in a component added for other purposes.

[0038] In this specification, when referring to a mixture of ingredients of a formulation, the term " Essentially composed of"It indicates that ingredients other than those explicitly listed may be present, but such ingredients are found only in trace amounts or in amounts low enough not to alter the basic characteristics of the formulation, including protein concentration, viscosity, thermal stability, osmotic pressure, and pH."

[0039] Term " polypeptide " and " protein " is used interchangeably to refer to a polymer of amino acid residues and is not limited to a minimum length. Such polymers of amino acid residues may include natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and fragments thereof are included in the definition. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, sialylation, acetylation, phosphorylation, etc. Additionally, for the purposes of the present invention, " polypeptide "Refers to a protein that includes modifications to its natural sequence, such as deletions, additions, and substitutions (which are generally virtually conserved), as long as the protein maintains the desired activity. Such modifications may be intentional through site-targeted mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification."

[0040] " FGFR2 " refers to human fibroblast growth factor receptor 2, including any selectively spliced ​​forms, e.g., IIIa, IIIb, and IIIc spliced ​​forms, unless non-human FGFR2 is explicitly indicated (e.g., "murine FGFR2"). The term FGFR2 includes wild-type FGFR2 and naturally occurring mutant forms, e.g., FGFR2 activating mutant forms, e.g., FGFR2-S252W found in some cancer cells. FGFR2 - IIIb" or " FGFR2b " is used interchangeably to refer to the fibroblast growth factor receptor 2 IIIb splice form. An exemplary human FGFR2-IIIb is denoted by GenBank accession number NP_075259.4 dated July 7, 2013. An exemplary non-restrictive mature human FGFR2-IIIb amino acid sequence is denoted by SEQ ID NO. 1. FGFR2 - IIIc " or " FGFR2c " is used interchangeably to refer to the fibroblast growth factor receptor 2 IIIc splice form. An exemplary human FGFR2-IIIc is denoted by GenBank accession number NP_000132.3 dated July 7, 2013. An exemplary non-restrictive mature FGFR2-IIIc amino acid sequence is denoted by SEQ ID NO. 13.

[0041] In relation to anti-FGFR2-IIIb antibodies, the term "ligand" The combination of Block " or ligand" The combination of suppress " refers to the ability to inhibit the interaction between FGFR2-IIIb and FGFR2 ligands, e.g., FGF1 or FGF2. Such inhibition may occur through any mechanism, including, for example, overlapping binding sites on FGFR2-IIIb and / or direct interference with ligand binding due to conformational changes of FGFR2-IIIb induced by antibodies that alter ligand affinity.

[0042] " Affinity " or " Binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). In some embodiments, "binding affinity" refers to the endogenous binding affinity reflecting the 1:1 interaction between the members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally given by the dissociation constant (K d It can be expressed by ).

[0043] Terms used in this specification " antibodies "..." refers to a molecule comprising at least heavy chain supervariable regions (HVR) H1, H2, and H3 and light chain supervariable regions (HVR) L1, L2, and L3, wherein the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, antibodies comprising full-length heavy chains and light chains, as well as fragments capable of binding to an antigen (also referred to herein as "antigen-binding fragments"), such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, such as mouse, human, cynomolgus monkey, etc. Antibodies also include other molecules, such as antibodies conjugated to small molecule drugs, heterospecific antibodies, and multispecific antibodies.

[0044] Term " Heavy chain variable area " refers to a region comprising heavy chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3. In some embodiments, the heavy chain variable region also includes at least a portion of FR1 and / or at least a portion of FR4.

[0045] Term " Heavy chain invariant region " is at least 3 heavy chain invariant domains, i.e., C H 1, C H 2, and C HRefers to a region containing 3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Additionally, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Specific isotypes may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region); IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region); IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0046] Term " Heavy chain " refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0047] Term " Light chain variable area " refers to a region comprising light chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also comprises FR1 and / or FR4.

[0048] Term " Light chain invariant region "is a light chain invariant domain, i.e., C LIt refers to a region containing . An exemplary light chain invariant region that is not restricted includes λ and κ.

[0049] Term " Light chain " refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, the light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0050] Term " Super variable area " or " HVR " refers to each region of an antibody variable domain that is hypervariable in the sequence and / or forms a structurally restricted loop ("hypervariable loop"). Generally, natural 4-chain antibodies contain 6 HVRs; V H 3 in (H1, H2, H3), V L There are three (L1, L2, L3). HVR generally contains amino acid residues derived from hypervariable loops and / or "complementary determination regions" (CDR), the latter having the highest sequence variability or being involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3). (Chothia and Lesk, J. Mol . Biol .196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24 to 34 of L1, amino acid residues 50 to 56 of L2, amino acid residues 89 to 97 of L3, amino acid residues 31 to 35B of H1, amino acid residues 50 to 65 of H2, and amino acid residues 95 to 102 of H3. (Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The terms hypervariable region (HVR) and complementarity determining region (CDR) both refer to parts of the variable region that form the antigen-binding region.

[0051] As used in this specification, " chimera antibodies " refers to an antibody comprising at least one variable region derived from a first species (e.g., mouse, rat, Philippine monkey, etc.) and at least one constant region derived from a second species (e.g., human, Philippine monkey, etc.). In some embodiments, the chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one Philippine monkey variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one rat variable region and at least one mouse constant region. In some embodiments, all variable regions of the chimeric antibody are derived from the first species and all constant regions of the chimeric antibody are derived from the second species.

[0052] As used in this specification, " Humanized antibodies" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region is replaced with a corresponding amino acid derived from a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is Fab, scFv, (Fab')2, etc.

[0053] As used in this specification, " CDR - Transplanted( grafted ) Antibodies " or " HVR - Transplanted antibodies " refers to a humanized antibody in which the complementarity determining region (CDR) or hypervariable region (HVR) of the first (non-human) species is transplanted into the framework region (FR) of the second (human) species.

[0054] As used in this specification, " Human antibodies " refers to antibodies produced in humans, antibodies produced in non-human animals containing human immunoglobulin genes, e.g., XenoMouse®, and antibodies selected using in vitro methods, e.g., phage display, wherein the antibody repertoire is based on human immunoglobulin sequences.

[0055] " Post-Foucauldron "Antibody or " Foucault Lacking "Antibody refers to an IgG1 or IgG3 isotype antibody lacking fuse in its constant domain glycosylation. Glycosylation of human IgG1 or IgG3 occurs in Asn297 (N297) as glycosylation of a core fucosylated biantennary complex oligosaccharide terminated by up to two Gal residues. In some embodiments, the defucosylated antibody lacks fuse in Asn297. These structures are designated as G0, G1 (α1,6 or α1,3), or G2 glycan residues depending on the amount of terminal Gal residues. For example, literature [Raju, TS, BioProcess Int Refer to . 1: 44-53 (2003). CHO-type glycosylation of antibody Fc, for example, in the literature [Routier, FH, Glycoconjugate J. It is described in [14: 201-207 (1997)]. Within an antibody population, if less than 5% of the antibodies in the population contain fucose from Asn297, the antibodies are considered to be defucosylated.

[0056] " Effector function "This refers to biological activity attributed to the Fc region of an antibody that varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation."

[0057] " Antibody-dependent cell-mediated cytotoxicity " or " ADCC " refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells [Ravetch and Kinet, Annu Rev. ImmunolIt is summarized in Table 3 on page 464 of [9:457-92 (1991)]. To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays may be performed, e.g., as described in U.S. Patent No. 5,500,362 or No. 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells useful for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, e.g., in the literature [Clynes et al . Proc . Natl . Acad . Sci . (USA) It can be evaluated in animal models as disclosed in [95:652-656 (1998)]. Additional antibodies having a modified Fc region amino acid sequence and increased or decreased ADCC activity are described, for example, in U.S. Patent No. 7,923,538 and U.S. Patent No. 7,994,290.

[0058] " Improved ADCC activeAn antibody having " is an antibody that is more effective in mediating ADCC in vitro or in vivo compared to a parent antibody when the amounts of the antibody and parent antibody used in the assay are essentially the same, wherein the antibody and the parent antibody differ in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is defucosylated while the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using an in vitro ADCC assay such as that disclosed in U.S. Publication No. 2015-0050273-A1, but other assays or methods for determining ADCC activity in animal models, for example, are considered. In other embodiments, the antibody with enhanced ADCC activity also has enhanced affinity for Fc gamma RIIIA. In some embodiments, the antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (V158). In certain embodiments, the antibody with enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA (F158).

[0059] " Fc gamma In RIIIA Improved affinity for"It refers to an antibody having a greater affinity for Fc gamma RIIIA (also referred to as CD16a in some examples) than a parent antibody, wherein the antibody and the parent antibody differ in at least one structural aspect. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is defucosylated while the parent antibody is fucosylated. Any suitable method for determining affinity for Fc gamma RIIIA may be used. In some embodiments, affinity for Fc gamma RIIIA is determined by the method described in U.S. Publication No. 2015-0050273-A1. In other embodiments, the antibody with enhanced affinity for Fc gamma RIIIA also has enhanced ADCC activity. In certain embodiments, the antibody with enhanced affinity for Fc gamma RIIIA has enhanced affinity for Fc gamma RIIIA (V158). In some embodiments, the antibody with enhanced affinity for Fc gamma RIIIA is Fc gamma It has enhanced affinity for RIIIA(F158).

[0060] Term " Leadership hierarchy "Leader sequence refers to a sequence of amino acid residues located at the N-terminus of a polypeptide that promotes the secretion of a polypeptide derived from a mammalian cell. The leader sequence can be cleaved upon release of the polypeptide from the mammalian cell to form a mature protein. The leader sequence may be natural or synthetic and may be heterogeneous or homologous to the attached protein. Non-limiting exemplary leader sequences include leader sequences derived from heterogeneous proteins. In some embodiments, the antibody lacks a leader sequence. In other embodiments, the antibody comprises at least one leader sequence, which may be selected from natural antibody leader sequences and heterogeneous leader sequences.

[0061] As used in this specification, the term " isolated"Isolated" refers to a molecule separated from at least some of the components typically found in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered "isolating" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" if it is not part of a larger polynucleotide typically found in nature (e.g., genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), or, for example, in the case of RNA polynucleotides, if the polynucleotide is separated from at least some of the components of the cell in which it was produced. Therefore, DNA polynucleotides contained in a vector inside a host cell may be referred to as "isolated" unless the polynucleotide is naturally found in that vector.

[0062] Term " object " and " patient " is used interchangeably to refer to humans in this specification. In some embodiments, methods for treating other mammals, including but not limited to rodents, primates, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets, are also provided.

[0063] Term " cancer " is used herein to refer to a group of cells exhibiting abnormally high levels of proliferation and growth. Cancer may be benign (also referred to as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid tumor cells or leukemia cancer cells. The term " Cancer growth"In this specification, it is used to refer to proliferation or growth by cells or cells containing cancer that causes a corresponding increase in the size or degree of cancer."

[0064] Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

[0065] As used in this specification, the term " therapy " refers to a therapeutic treatment intended, for example, to cure a condition or disorder, to reduce the severity of a condition or disorder, to slow its progression, or to prevent the recurrence of a condition or disorder. In certain embodiments, the term " therapy "includes any administration or application of a therapeutic agent for a disease in a patient, and includes inhibiting or slowing the disease or the progression of the disease; partially or completely alleviating the disease by, for example, inducing degeneration, or restoring or recovering functional loss, loss, or defect; stimulating inefficient processes; or reducing severity by inducing a disease plateau. The term " therapyIt also includes reducing the severity of any phenotypic feature and / or reducing the incidence, degree, or likelihood of such feature. Persons requiring treatment include not only those who already have a disability, but also those at risk of recurrence of the disability or those for whom recurrence of the disability must be prevented or delayed.

[0066] Term " Effective amount " or " Therapeutic effective dose "Effective amount" refers to an amount of drug effective for treating a disease or disorder in a subject. In certain embodiments, an effective amount refers to the dosage required to achieve a desired therapeutic result and an effective amount over a period of time. The therapeutically effective amount of the anti-FGFR2 antibody of the present invention may vary depending on factors such as the individual's disease state, age, sex, and body weight, and the ability of the antibody or antibodies to induce a desired response in the individual. The therapeutically effective amount comprises an amount in which the therapeutically beneficial effect outweighs any toxic or harmful effect of the antibody or antibodies. In some embodiments, the expression "effective amount" refers to an amount of antibody effective for treating cancer.

[0067] Additional definitions are provided in the following section.

[0068] port- FGFR2 antibodies

[0069] In any formulation described herein containing an anti-FGFR2 antibody, the anti-FGFR2 antibody may be a monoclonal antibody, a genetically engineered antibody, a humanized antibody, a chimeric antibody, or a human antibody. In any composition or method described herein, the anti-FGFR2 antibody may be selected from Fab, Fv, scFv, Fab', and (Fab')2. In any composition or formulation described herein, the anti-FGFR2 antibody may be selected from IgA, IgG, and IgD. In any composition or formulation described herein, the anti-FGFR2 antibody may be IgG. In any method described herein, the antibody may be IgG1 or IgG3.

[0070] An exemplary anti-FGFR2 antibody comprises an antibody that binds to FGFR2-IIIb. In some embodiments, the anti-FGFR2-IIIb antibody binds to FGFR2-IIIc with a lower affinity than that which binds to FGFR2-IIIb. In other embodiments, the anti-FGFR2-IIIb antibody does not bind detectably to FGFR2-IIIc.

[0071] An exemplary anti-FGFR2-IIIb antibody for use in the embodiments of this specification is the HuGAL-FR21 antibody described in U.S. Patent No. 8,101,723 B2, issued January 24, 2012, which is specifically incorporated herein by reference. Figures 13 and 14 of U.S. Patent No. 8,101,723 B2 illustrate the amino acid sequences of the variable region and the full-length mature antibody chain of HuGAL-FR21, which are incorporated herein by reference. The sequence of the heavy chain variable region of the antibody HuGAL-FR21 is underlined in Figure 13 of U.S. Patent No. 8,101,723 B2, which is specifically incorporated herein by reference. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297. Additional antibodies that may be used in the embodiments of this specification include those described in U.S. Patent Publication No. 2015-0050273-A1, which describe a specific defucosylated FGFR2-IIIb antibody, which is incorporated herein by reference.

[0072] In some embodiments, the anti-FGFR2-IIIb antibody comprises at least 1, 2, 3, 4, 5, or 6 hypervariable regions (HVR; e.g., CDR) selected from (a) HVR-H1 having the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 having the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 having the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 having the amino acid sequence of SEQ ID NO. 9; (e) HVR-L2 having the amino acid sequence of SEQ ID NO. 10; and (f) HVR-L3 having the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0073] In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the anti-FGFR2-IIIb antibody comprises at least one heavy chain comprising at least a portion of a heavy chain variable region and a heavy chain constant region, and at least one light chain comprising at least a portion of a light chain variable region and a light chain constant region. In some embodiments, the anti-FGFR2-IIIb antibody comprises two heavy chains (wherein each heavy chain comprises at least a portion of a heavy chain variable region and a heavy chain constant region), and two light chains (wherein each light chain comprises at least a portion of a light chain variable region and a light chain constant region). In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 5. In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 3. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0074] In some embodiments, the anti-FGFR2-IIIb antibody comprises six HVRs comprising (a) HVR-H1 having the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 having the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 having the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 having the amino acid sequence of SEQ ID NO. 9; (e) HVR-L2 having the amino acid sequence of SEQ ID NO. 10; and (f) HVR-L3 having the amino acid sequence of SEQ ID NO. 11. In some embodiments, the anti-FGFR2-IIIb antibody comprises six HVRs as described above and binds to FGFR2-IIIb. In some embodiments, the anti-FGFR-IIIb antibody does not bind to FGFR2-IIIc. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0075] In one embodiment, the anti-FGFR2-IIIb antibody competes with six HVRs comprising (a) HVR-H1 having the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 having the amino acid sequence of SEQ ID NO. 7; (c) HVR-H3 having the amino acid sequence of SEQ ID NO. 8; (d) HVR-L1 having the amino acid sequence of SEQ ID NO. 9; (e) HVR-L2 having the amino acid sequence of SEQ ID NO. 10; and (f) HVR-L3 having the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0076] In some embodiments, the anti-FGFR2-IIIb antibody comprises at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 having the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 having the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 having the amino acid sequence of SEQ ID NO. 8. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose in Asn297.

[0077] In some embodiments, the anti-FGFR2-IIIb antibody comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 having the amino acid sequence of SEQ ID NO. 9; (b) HVR-L2 having the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 having the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0078] In some embodiments, the anti-FGFR2-IIIb antibody comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0079] In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 4. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, but the anti-FGFR2-IIIb antibody comprising such sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such an FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectably binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from SEQ ID NO. 4. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR). Optionally, the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO. 5, including post-translational modifications of the corresponding sequence. In certain embodiments, the VH comprises 1, 2, or 3 HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0080] In some embodiments, the anti-FGFR2-IIIb antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 5. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, but the anti-FGFR2-IIIb antibody comprising said sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, the anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted from SEQ ID NO. 5. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR). Optionally, the anti-FGFR2-IIIb antibody comprises the VL sequence of SEQ ID NO. 4, including post-translational modifications of the corresponding sequence. In certain embodiments, the VL comprises 1, 2, or 3 HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0081] In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 4, and a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 5. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, and a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, but an anti-FGFR2-IIIb antibody containing said sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 4. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 5. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR). Optionally, the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO. 4 and the VL sequence of SEQ ID NO. 5, including post-translational modifications of one or two sequences.In certain embodiments, VH comprises 1, 2, or 3 HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8; and VL comprises 1, 2, or 3 HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0082] In some embodiments, the anti-FGFR2-IIIb antibody comprises VH as in any of the embodiments provided above, and VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO. 4 and SEQ ID NO. 5, respectively, including post-translational modifications of the corresponding sequences. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0083] In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 2. In certain embodiments, the heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, but the anti-FGFR2-IIIb antibody comprising said sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such anti-FGFR2-IIIb antibodies retain the ability to selectively bind to FGFR2-IIIb without detectably binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 2. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR). Optionally, the anti-FGFR2-IIIb antibody comprises the VH sequence of SEQ ID NO. 2, including post-translational modifications of the corresponding sequence. In certain embodiments, the heavy chain comprises 1, 2, or 3 HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0084] In some embodiments, the anti-FGFR2-IIIb antibody comprises a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 3. In certain embodiments, the light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion with respect to the reference sequence, but the anti-FGFR2-IIIb antibody comprising said sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such anti-FGFR2-IIIb antibodies retain the ability to selectively bind to FGFR2-IIIb without detectably binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 3. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR). Optionally, the anti-FGFR2-IIIb antibody comprises the VL sequence of SEQ ID NO. 3, including post-translational modifications of the corresponding sequence. In certain embodiments, the light chain comprises 1, 2, or 3 HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0085] In some embodiments, the anti-FGFR2-IIIb antibody comprises a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 2 and a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO. 3. In certain embodiments, a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a reference sequence comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion, but an anti-FGFR2-IIIb antibody containing such a sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectably binding to FGFR2-IIIc. In certain embodiments, a light chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence comprises a substitution (e.g., a conservative substitution), an insertion, or a deletion, but an anti-FGFR2-IIIb antibody containing such a sequence retains the ability to bind to FGFR2-IIIb. In certain embodiments, such an anti-FGFR2-IIIb antibody retains the ability to selectively bind to FGFR2-IIIb without detectably binding to FGFR2-IIIc. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 2. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO. 3. In certain embodiments, the substitution, insertion, or deletion occurs in the outer region of HVR (i.e., in FR).Optionally, the anti-FGFR2-IIIb antibody heavy chain comprises the VH sequence of SEQ ID NO. 2, including a post-translational modification of the corresponding sequence, and the anti-FGFR2-IIIb antibody light chain comprises the VL sequence of SEQ ID NO. 3, including a post-translational modification of the corresponding sequence. In certain embodiments, the heavy chain comprises 1, 2, or 3 HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 7; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 8; and the light chain comprises 1, 2, or 3 HVRs selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 10; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 11. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0086] Additional exemplary anti-FGFR2 antibodies are the GAL-FR22 and GAL-FR23 antibodies described in U.S. Patent No. 8,101,723 B2, incorporated herein by reference. For example, the light and heavy chain variable regions of GAL-FR22 are provided as SEQ ID NOs. 7 and 8 in Patent No. 8,101,723 B2, while the Kabat CDR and light and heavy chain variable regions are also provided in FIG. 16 of the said patent, which is incorporated herein by reference. Hybridomas producing GAL-FR21, GAL-FR22, and GAL-FR23 were deposited in the American Type Culture Collection (American Type Culture Collection; PO Box 1549, Manassas, Virginia, USA 20108) on November 6, November 6, and August 12, 2008, respectively, under ATCC numbers 9586, 9587, and 9408. Accordingly, in some embodiments, the anti-FGFR2 antibody is an antibody comprising the amino acid sequence of an antibody obtained from one of these three hybridoma strains.

[0087] The heavy chain and light chain variable regions of GAL-FR22 are also presented herein as SEQ ID NOs 15 and 19, while the Kavat CDR is presented herein as SEQ ID NOs 16 to 19 and 20 to 22. Accordingly, in some embodiments, the anti-FGFR2-IIIb antibody heavy chain variable region comprises (i) HVR1 (CDR1) comprising the amino acid sequence of SEQ ID NO. 16; (ii) HVR2 comprising the amino acid sequence of SEQ ID NO. 17; and (iii) HVR3 comprising the amino acid sequence of SEQ ID NO. 18; and the light chain variable region comprises (iv) HVR1 comprising the amino acid sequence of SEQ ID NO. 20; (v) HVR2 comprising the amino acid sequence of SEQ ID NO. 21; and (vi) HVR3 comprising the amino acid sequence of SEQ ID NO. 22.

[0088] In some embodiments, the anti-FGFR2 antibody comprises an FGFR2-IIIb antibody comprising the amino acid sequence of SEQ ID NO. 15, wherein the heavy chain variable domain is at least 95% identical, e.g., at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO. 15. In some embodiments, the anti-FGFR2 antibody comprises an FGFR2-IIIb antibody comprising the amino acid sequence of SEQ ID NO. 19, wherein the light chain variable domain is at least 95% identical, e.g., at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO. 19. In some embodiments, the heavy chain variable domain is at least 95% identical to the amino acid sequence of SEQ ID NO. 15, e.g., at least 97%, at least 98%, or at least 99%, or comprises the amino acid sequence of SEQ ID NO. 39, and the light chain variable domain is at least 95% identical to the amino acid sequence of SEQ ID NO. 19, e.g., at least 97%, at least 98%, or at least 99%, or comprises the amino acid sequence of SEQ ID NO. 19. In some such embodiments, the anti-FGFR2-IIIb antibody heavy chain variable domain also comprises (i) HVR1 (CDR1) comprising the amino acid sequence of SEQ ID NO. 16; (ii) HVR2 comprising the amino acid sequence of SEQ ID NO. 17; and (iii) HVR3 comprising the amino acid sequence of SEQ ID NO. 18; and / or; the light chain variable domain also comprises (iv) HVR1 comprising the amino acid sequence of SEQ ID NO. 20; (v) HVR2 comprising the amino acid sequence of SEQ ID NO. 21; and (vi) include HVR3 comprising the amino acid sequence of SEQ ID NO. 22. In some embodiments, the antibody is an IgG1 or IgG3 antibody lacking fucose from Asn297.

[0089] Post-Foucauldron port- FGFR2 antibodies

[0090] In some embodiments, the anti-FGFR2 antibody described herein has a fucose-deficient carbohydrate structure attached (directly or indirectly) to the Fc region, i.e., the antibody is defucosylated. In some embodiments, the defucosylated antibody is an IgG1 or IgG3 antibody defucosylated from Asn297.

[0091] In this specification, an antibody is considered "defucosylated" when a plurality of such antibodies comprises at least 95% defucosylated antibodies. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chains in Asn297 against the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures). Non-limiting exemplary methods for detecting fucose in antibodies include MALDI-TOF mass spectrometry (see, e.g., WO 2008 / 077546), HPLC measurement of emitted fluorescence-labeled oligosaccharides (e.g., Schneider et al., "N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection," Agilent Technologies, Inc. (2012)); [Lines, J. Pharm . Biomed .Analysis , 14: 601-608 (1996)]; [Takahasi, J. Chrom [Refer to ., 720: 217-225 (1996)]), capillary electrophoresis of emitted fluorescence-labeled oligosaccharides (e.g., [Ma et al., Anal. Chem . [See , 71: 5185-5192 (1999)]), and HPLC using detection by pulse amperometrics to measure monosaccharide composition (e.g., literature [Hardy, et al., Analytical Biochem.Includes [see , 170: 54-62 (1988)]. In some embodiments, in the composition of the defucosylated antibody of this specification, fucose cannot be detected by one or more of these methods.

[0092] Asn297 refers to an asparagine residue located at position approximately 297 in the Fc region (EU numbering of Fc region residues); however, in a given antibody sequence, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to small sequence variations of the antibody. In the FGFR2-IIIb antibody described herein, Asn297 is sequence QY N It is found in ST and is underlined in bold at sequence number 2 of the sequence table shown below.

[0093] Fucosylated variants may have improved ADCC functionality. For example, US Patent Publication US 2003 / 0157108 (Presta, L.); Refer to US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). For examples of publications related to "defucosylated" or "fucose-deficient" antibodies, see US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Literature[Okazaki et al. J. Mol . Biol . 336:1239-1249 (2004)]; literature[Yamane-Ohnuki et al. Biotech. BioengIncludes . 87: 614 (2004)]. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Literature [Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986)]; US patent application US 2003 / 0157108 A1 (Presta, L.); and WO 2004 / 056312 A1 (Adams et al .), particularly Example 11), and knockout cell lines, e.g., cell lines deficient in the functional alpha-1,6-fucosyltransferase gene FUT8, e.g., knockout CHO cells (e.g., literature [Yamane-Ohnuki] et al. Biotech. Bioeng. 87: 614 (2004)]; [Kanda, Y. et al., Biotechnol. Bioeng. Includes , 94(4):680-688 (2006)]; and WO2003 / 085107).

[0094] The anti-FGFR2 antibodies of this specification may also have a bisected oligosaccharide, for example, in which a two-antenna-type oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are, for example, WO 2003 / 011878 (Jean-Mairet et al .); US Patent 6,602,684(Umana et al .); and US 2005 / 0123546(Umana et al It is described in .). In some embodiments, the FGFR2 antibody has at least one galactose residue in the oligosaccharide attached to the Fc region. Such an antibody may have an enhanced CDC function. Such an antibody is, for example, WO 1997 / 30087 (Patel et al It is described in WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0095] In a specific embodiment of the present invention, a defucosylated FGFR2 antibody mediates ADCC more effectively in the presence of human effector cells than an antibody having the same amino acid sequence containing fucose. Generally, ADCC activity can be determined using the in vitro ADCC assay disclosed in U.S. Publication 2015-0050273 A1, but other assays or methods for determining ADCC activity are considered, for example, in animal models.

[0096] In some embodiments, the FGFR2 antibody comprises the heavy chain and light chain sequences of SEQ ID NOs 2 and 3. In other embodiments, the antibody comprising the heavy chain and light chain sequences of SEQ ID NOs 2 and 3 is defucosylated.

[0097] Pharmaceutical formulation of anti-FGFR2 antibody

[0098] The present disclosure provides a pharmaceutical formulation of an anti-FGFR2 antibody suitable for administration to humans. The anti-FGFR2 antibody may be any such antibody disclosed herein. The amount and type of composition in the pharmaceutical formulation are selected to provide maximum stability for the antibody and thus for maximum shelf life.

[0099] A pharmaceutical formulation may comprise, for example, an anti-FGFR2 antibody; a buffer selected from histidine, citrate, and phosphate; sucrose; and a surfactant. In some embodiments, the formulation essentially consists of an anti-FGFR2 antibody; a buffer selected from histidine, citrate, and phosphate; sucrose; and a surfactant. Alternatively, the pharmaceutical formulation may comprise, for example, an anti-FGFR2 antibody; a buffer selected from histidine, citrate, and phosphate; arginine; and a surfactant. In some embodiments, the formulation essentially consists of an anti-FGFR2 antibody; a buffer selected from histidine, citrate, and phosphate; arginine; and a surfactant. The pharmaceutical formulation may comprise, for example, an anti-FGFR2 antibody, histidine, sucrose, and a surfactant. In some embodiments, the formulation essentially consists of an anti-FGFR2 antibody, histidine, sucrose, and a surfactant. Alternatively, the pharmaceutical formulation may comprise, for example, an anti-FGFR2 antibody, histidine, arginine, and a surfactant. In some embodiments, the formulation essentially consists of an anti-FGFR2 antibody, histidine, arginine, and a surfactant. In some embodiments, the formulation is suitable for intravenous administration to a patient, for example, by infusion or injection. In other embodiments, the formulation is suitable for subcutaneous administration to a patient, for example, by injection. In certain embodiments, the formulation is isotonic with the body fluid of the administration site, for example, human plasma or lymph fluid.

[0100] In some embodiments, the anti-FGFR2 antibody of this specification was found to be soluble in aqueous formulations up to 180 mg / ml. In some embodiments, the formulation is 10 to 180 mg / ml anti-FGFR2 antibody, or 10 to 30 mg / ml anti-FGFR2 antibody, or, for example, 10 to 25 mg / ml, 20 to 30 mg / ml, 15 to 25 mg / ml, 10 to 15 mg / ml, 15 to 20 mg / ml, 20 to 25 mg / ml, 18 to 22 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 It contains anti-FGFR2 antibodies of mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, or 30 mg / ml. In some embodiments, the formulation contains 20 mg / ml of antibody.

[0101] In some embodiments, the formulation comprises histidine, citrate, or phosphate as a buffer. In some such cases, the formulation buffer is essentially histidine. In some embodiments, the formulation comprises 5 to 40 mM of histidine, citrate, or phosphate, e.g., 10 to 40 mM, 10 to 30 mM, 15 to 25 mM, 10 to 20 mM, 20 to 30 mM, 15 to 20 mM, 20 to 25 mM, 25 to 30 mM, or 18 to 22 mM. In some embodiments, the formulation comprises a range limited to 10 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM histidine, citrate, or phosphate, or two of these concentrations. In certain embodiments, the formulation comprises 20 mM histidine, citrate, or phosphate.

[0102] In some embodiments, the formulation comprises histidine as a buffer. In some such embodiments, the formulation does not comprise citrate or phosphate. In some such cases, the formulation buffer essentially consists of histidine. In some embodiments, the formulation comprises citrate as a buffer. In some such embodiments, the formulation does not comprise histidine or phosphate. In some such cases, the formulation buffer essentially consists of citrate. In some embodiments, the formulation comprises phosphate as a buffer. In some such embodiments, the formulation does not comprise citrate or histidine. In some such cases, the formulation buffer essentially consists of phosphate.

[0103] In some embodiments comprising or essentially composed of histidine as a buffer, the formulation comprises 5 to 40 mM histidine, e.g., 10 to 40 mM, 10 to 30 mM, 15 to 25 mM, 10 to 20 mM, 20 to 30 mM, 15 to 20 mM, 20 to 25 mM, 25 to 30 mM, or 18 to 22 mM histidine. In some embodiments, the formulation comprises 10 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM histidine, or a range limited to two of these concentrations. In some embodiments, the formulation comprises 20 mM histidine.

[0104] In some embodiments, the surfactant is a polysorbate, e.g., polysorbate 20 or polysorbate 80. In some such cases, the formulation comprises 0.001 to 0.1%, 0.002 to 0.1%, 0.01 to 0.1%, 0.005% to 0.05%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 20 or 80. In some embodiments, the formulation comprises 0.005% to 0.05% polysorbate 20, e.g., 0.008% to 0.012% polysorbate 20. In other embodiments, the formulation comprises 0.002% to 0.1% polysorbate 20. In some embodiments, the formulation comprises 0.01% polysorbate 20.

[0105] Sucrose formulations including

[0106] In some embodiments, the formulation comprises or essentially consists of a mixture of an anti-FGFR2 antibody, histidine, a polysorbate (e.g., polysorbate 20), and sucrose. The concentrations of the antibody, histidine, and polysorbate may be, for example, as provided above. In some embodiments, sucrose may be found in an amount that provides a formulation isotonic with body fluids. In some embodiments, the formulation comprises 200 to 300 mM sucrose, e.g., 200 to 250 mM, 250 to 300 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM. In some embodiments, the formulation comprises 250 to 300 mM sucrose. In another embodiment, the formulation comprises 260 to 280 mM sucrose. In certain embodiments, the formulation comprises 250 to 270 mM sucrose. In some embodiments, the formulation comprises 270 to 300 mM sucrose. In other embodiments, the formulation comprises 250 mM, 260 mM, 270 mM, or 280 mM sucrose. In certain embodiments, the formulation comprises 270 mM sucrose. In some embodiments, the formulation comprising sucrose does not contain arginine.

[0107] In some embodiments, the pH range of the pharmaceutical formulation is 5.0 to 7.0. In some embodiments, the sucrose-based pharmaceutical formulation has a pH of 5.0 to 6.5, e.g., 5.0 to 6.0, 5.5 to 6.0, 5.5 to 6.5, 5.8 to 6.2, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH is 5.8 to 6.2. In some embodiments, the pH is 6.0.

[0108] Formulations containing arginine

[0109] In some embodiments, the formulation comprises histidine, arginine, a polysorbate (e.g., polysorbate 20), and an antibody, or is essentially composed of these. In some such embodiments, the formulation does not contain sucrose.

[0110] In some embodiments, arginine is found at 100 to 200 mM, e.g., 100 to 150 mM, 150 to 200 mM, 130 to 170 mM, or 140 to 160 mM. In certain embodiments, arginine is present at concentrations of 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM. In other embodiments, arginine is found at 140 mM, 150 mM, or 160 mM. In some embodiments, arginine is present at 150 mM.

[0111] In some embodiments, the pH range of the pharmaceutical formulation is 5.0 to 7.0. In some embodiments, the arginine-based pharmaceutical formulation has a pH of 5.0 to 6.5, e.g., 5.0 to 6.0, 5.5 to 6.5, 5.5 to 6.0, 5.5 to 5.9, 5.6 to 5.8, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. In some embodiments, the pH is 5.5 to 5.9. In some embodiments, the pH is 5.6 to 5.8. In some embodiments, the pH is 5.7.

[0112] Specific formulation embodiments

[0113] In another embodiment, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.002 to 0.1% polysorbate 20, and 10 to 30 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.0 to 7.0. In some such cases, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.002 to 0.1% polysorbate 20, and one or more buffers selected from 10 to 30 mM histidine, citrate, and phosphate, wherein the formulation has a pH of 5.0 to 7.0.

[0114] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.005 to 0.05% polysorbate 20, and 10 to 30 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical formulation essentially comprises 10 to 30 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.005 to 0.05% polysorbate 20, and one or more buffers selected from 10 to 30 mM histidine, citrate, and phosphate, wherein the formulation has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.005 to 0.05% polysorbate 20, and 10 to 20 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical formulation essentially comprises 10 to 20 mg / ml anti-FGFR2 antibody, 130 to 170 mM arginine or 250 to 290 mM sucrose, 0.005 to 0.05% polysorbate 20, and 10 to 30 mM histidine, citrate, and phosphate, with one or more buffers selected from these, wherein the formulation has a pH of 5.0 to 6.5.

[0115] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 140 to 160 mM arginine or 260 to 280 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially comprises 10 to 30 mg / ml anti-FGFR2 antibody, 140 to 160 mM arginine or 260 to 280 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 140 to 160 mM arginine or 260 to 280 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially comprises 10 to 20 mg / ml anti-FGFR2 antibody, 140 to 160 mM arginine or 260 to 280 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5.

[0116] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM of histidine, citrate, and phosphate, wherein the formulation has a pH of 5.5 to 6.5.

[0117] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.005 to 0.05% polysorbate 20, and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5.

[0118] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20, and 10 to 30 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20, and 10 to 30 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 10 to 30 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 10 to 30 mM histidine, wherein the formulation has a pH of 5.5 to 6.5.

[0119] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.008 to 0.012% polysorbate 20 and 15 to 25 mM histidine, wherein the formulation has a pH of 5.5 to 6.5.

[0120] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.01% polysorbate 20 and 20 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.01% polysorbate 20 and 20 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.01% polysorbate 20 and 20 mM histidine, wherein the formulation has a pH of 5.5 to 6.5. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine or 270 mM sucrose, 0.01% polysorbate 20 and 20 mM histidine, wherein the formulation has a pH of 5.5 to 6.5.

[0121] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.6 to 5.8 or 5.7. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 150 mM arginine, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.6 to 5.8 or 5.7. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.6 to 5.8 or 5.7. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 150 mM arginine, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.6 to 5.8 or 5.7.

[0122] In some embodiments, the pharmaceutical formulation comprises 10 to 30 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.9 to 6.1 or 6.0. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.9 to 6.1 or 6.0. In some embodiments, the pharmaceutical formulation comprises 10 to 20 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.9 to 6.1 or 6.0. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 20 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.9 to 6.1 or 6.0.

[0123] In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.0 to 6.0. In some embodiments, the pharmaceutical formulation essentially consists of 10 to 30 mg / ml anti-FGFR2 antibody, 270 mM sucrose, 0.01% polysorbate 20, and 20 mM histidine, wherein the formulation has a pH of 5.5 to 6.0.

[0124] In some embodiments, the formulation does not contain certain types of excipients. For example, in some embodiments, the formulation does not contain one or more of sugar alcohols, proteins other than anti-FGFR2 antibodies, surfactants other than polysorbates, and amino acids other than arginine and / or histidine. In certain embodiments containing arginine, the formulation does not contain one or more of sugars, sugars, sugar alcohols, proteins other than anti-FGFR2 antibodies, surfactants other than polysorbates, and amino acids other than arginine and histidine. In some embodiments containing sucrose, the formulation does not contain one or more of sugars other than sucrose, sugar alcohols, proteins other than anti-FGFR2 antibodies, surfactants other than polysorbates, and amino acids other than histidine. In some embodiments containing arginine, the formulation does not contain any of sugars, sugar alcohols, proteins other than anti-FGFR2 antibodies, surfactants other than polysorbates, and amino acids other than arginine and histidine. In some embodiments comprising sucrose, the formulation does not contain any of the following: sugars other than sucrose, sugar alcohols, proteins other than anti-FGFR2 antibodies, surfactants other than polysorbates, and amino acids other than histidine. In certain embodiments, the formulation does not contain any other buffering components other than histidine, citrate, and / or phosphate. In certain embodiments comprising histidine, citrate, or phosphate as a buffer, the formulation does not contain any other buffering components. As previously noted in this application, the expression “does not contain” in this context means that the excluded component is not present above a trace level, for example, due to contaminants or impurities found in intentionally added components.

[0125] Exemplary characteristics of the formulation

[0126] In some embodiments, the formulation is stored as a liquid and is not lyophilized before administration to a patient. In some embodiments, the formulation is an immediately available liquid formulation and can therefore be administered directly to a patient. Liquid formulations offer the advantage of being easier to administer to a patient and being immediately available than lyophilized formulations. In other embodiments, the formulation is diluted with saline or water, or mixed with other substances to dilute the protein before administration. For example, a formulation designed for IV infusion may be diluted with saline or other suitable buffer in an IV bag before administration. In some embodiments, the liquid formulation, e.g., an immediately available liquid formulation, is administered directly intravenously without dilution.

[0127] In some embodiments, the formulation is administered intravenously, for example, by intravenous (IV) infusion. Administration may take place in a hospital, clinic, outpatient, or other medical office setting. IV administration allows for the administration of the protein therapeutic agent in a relatively large amount of liquid.

[0128] In other embodiments, for higher protein (antibody) concentrations (e.g., greater than 300 mg / mL), the pharmaceutical formulations of this specification are injectable or administered subcutaneously, for example, by injection using a subcutaneous administration device. In some embodiments, the formulation is contained in a vial. In other embodiments, the vial is part of or attached to a subcutaneous administration device, e.g., a syringe and needle. In some embodiments, the vial is a single-use vial. In this specification, the vial may contain formulations of, for example, 0.5 mL, 1 mL, 1.5 mL, 2 mL, or 3 mL. In some embodiments, a complete single dose of the anti-FGFR2 antibody may be administered in one, two, or three subcutaneous administrations. Thus, in some such embodiments, a complete single dose of the antibody may be contained in one, two, or three single-use vials. In certain embodiments, the vial contains a formulation of 1 to 2 mL. Accordingly, in some embodiments, a complete single dose for a patient may be contained in a single 1 ml, 1.5 ml, 2 ml, 2.5 ml, or 3 ml vial or in two or three 0.5 ml, 1 ml, 0.5 ml, or 2 ml vials. In some embodiments, the liquid formulation is stored in the dark, for example, in a vial in the dark.

[0129] In some embodiments, the pharmaceutical formulation of this specification remains stable in solution and is suitable for pharmaceutical use, for example, after 3 or 6 months at 40°C. In other embodiments, the pharmaceutical formulation of this specification remains stable in solution and is suitable for pharmaceutical use, for example, after 6 months at 5°C and / or 25°C. In certain embodiments, protein aggregation in the formulation may increase by 3%, 4%, 5%, 6%, or 7% or less after 1 month, 2 months, or 3 months of storage at 40°C. In yet another embodiment, protein aggregation in the formulation may increase by 3% or 4% or less after 1 month of storage at 40°C and / or by 7% or less after 3 months of storage at 40°C. In some embodiments, protein aggregation in the formulation may increase by 2% or 2.5% or less after 3 months of storage at 25°C. In some embodiments, protein aggregation in the formulation may increase to 2% or less after storage at 5°C for 6 months.

[0130] In addition to aggregates, charged variants of the protein may be formed during storage as a result of chemical changes, such as deamidation and oxidation. For example, in some embodiments, the acidic variant of the anti-FGFR2 antibody does not increase or decrease by more than 20% after 3 months of storage at 25°C. In some embodiments, there is no change in the percentage of charged variants after 6 months of storage at 5°C. The appearance of charged variants can be detected, for example, using cation- or anion-exchange chromatography.

[0131] In certain embodiments, the pharmaceutical composition comprises 40% or less of an acidic variant of the anti-FGFR2 antibody and 20% or less of a basic variant of the anti-FGFR2 antibody after storage at 40°C for one month. In some embodiments, the pharmaceutical composition comprises 1% to 40%, 15% to 40%, or 20% to 40%, 20% to 30%, or 30% to 40% of an acidic variant of the antibody and / or 5% to 30%, 5% to 20%, 10% to 20%, 20% to 30%, or 15% to 20% of a basic variant of the antibody after one month at 40°C. In some embodiments, the pharmaceutical composition comprises an anti-FGFR2 antibody, wherein the composition comprises an acidic variant of the antibody in an amount of 15% to 35%, 20% to 30%, or 25% to 30% after 3 months at 25°C, and / or a basic variant of the antibody in an amount of 15% to 40%, 15% to 30%, 15% to 25%, 15% to 20%, or 20% to 25%, or an antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises an anti-FGFR2 antibody, wherein the composition comprises an acidic variant of the antibody in an amount of 15% to 30%, 15% to 25%, 20% to 30%, or 20% to 25% after 6 months at 5°C, and / or a basic variant of the antibody in an amount of 20% to 35%, 20% to 30%, or 25% to 30%, or an antigen-binding fragment thereof.

[0132] For example, in some embodiments, a formulation comprising histidine, sucrose, and polysorbate 20 at pH 5.5 to 6.5 in this specification initially comprises 20 to 25% of an acidic variant of the anti-FGFR2 antibody, while after storage at 40°C for 1 month, it comprises 20% to 40% of an acidic variant, after storage at 25°C for 3 months, it comprises 25% to 30% of an acidic variant, and after storage at 5°C for 6 months, it comprises 20% to 25% of an acidic variant. (See FIG. 27A to FIG. 27C.) In some embodiments, a formulation comprising histidine, arginine, and polysorbate 20 at pH 5.5 to 6.5 in this specification initially comprises 20 to 25% acidic variants of anti-FGFR2 antibody, while after storage at 40°C for 1 month, it comprises 20% to 40%, e.g., 30% to 40% acidic variants, after storage at 25°C for 3 months, it comprises 25% to 30% acidic variants, and after storage at 5°C for 6 months, it comprises 20% to 25% acidic variants. (See FIG. 28A to 28C.) In some embodiments, a formulation comprising histidine, sucrose, and polysorbate 20 at pH 5.5 to 6.5 in this specification initially comprises about 30% of a basic variant of the anti-FGFR2 antibody, while after storage at 40°C for 1 month, it comprises 10% to 25% of a basic variant, after storage at 25°C for 3 months, it comprises 15% to 30%, e.g., 15% to 25% of a basic variant, and after storage at 5°C for 6 months, it comprises 25% to 30% of a basic variant. (See FIG. 29A to 29C.) In some embodiments, pH 5.5 to 6 in this specification.The formulation comprising histidine, arginine, and polysorbate 20 in 5 initially contains 30% to 35% of a basic variant of the anti-FGFR2 antibody, while after storage at 40°C for 1 month, it contains 10% to 20% of a basic variant, after storage at 25°C for 3 months, it contains 15% to 30%, e.g., 15% to 25% of a basic variant, and after storage at 5°C for 6 months, it contains 25% to 30% of a basic variant. (See FIGS. 30A to 30C.)

[0133] In some embodiments, the pharmaceutical formulation remains stable for pharmaceutical use after one or more freeze-thaw cycles, e.g., 2, 3, 4, or 5 freeze-thaw cycles, at -70°C. For example, in some embodiments, protein aggregation in the formulation may increase by 2.0%, 1.5%, 1.0%, or 0.5% or less after 5 freeze-thaw cycles. In certain embodiments, the percentage of high molecular weight protein species, as measured by light scattering, remains unchanged or increases by 0.5% or less after 5 freeze-thaw cycles. In some embodiments, the formulation also remains stable against mechanical stress, which can be determined by exposing a vial containing the formulation to stirring for an extended period. In other embodiments, aggregation may increase by 2.0%, 1.5%, 1.0%, or 0.5% or less after 72 hours of mechanical stress at 500 rpm.

[0134] Treatment method using an anti-FGFR2 antibody pharmaceutical formulation

[0135] The pharmaceutical formulations disclosed herein may be used in a method for treating patients requiring anti-FGFR2 antibody therapy. The use of anti-FGFR2 antibodies is disclosed, for example, in international patent publications WO2015 / 017600 and WO2017 / 091577, which are respectively incorporated herein by reference.

[0136] In some embodiments, the pharmaceutical formulations of this specification may be used to treat cancer patients. In some embodiments, the cancer is a solid tumor. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous non-small cell lung cancer), lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head and neck cancer (including squamous cell carcinoma of the head and neck). In some embodiments, the cancer is gastric cancer or bladder cancer.

[0137] In some embodiments, the cancer is relapsed or advanced after surgery, chemotherapy, radiation therapy, or a combination thereof. In some embodiments, the subject is an insufficient responder to PD-1 / PD-L1 inhibitors. A subject who is an insufficient responder to PD-1 / PD-L1 inhibitors is a subject who may have previously responded to PD-1 / PD-L1 inhibitors but may become less responsive to PD-1 / PD-L1 inhibitors, or a subject who may never have responded to PD-1 / PD-L1 inhibitors. In some embodiments, the subject has previously received PD-1 / PD-L1 inhibitor therapy.

[0138] In some embodiments, the cancer comprises FGFR2 gene amplification. In some embodiments, the cancer comprising FGFR2 gene amplification also overexpresses FGFR2IIIb. In some embodiments, the cancer comprising FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the cancer comprising FGFR2 amplification expresses FGFR2IIIb at a normalization level greater than 2, 3, 5, or 10 times the normalization level of FGFR2IIIc expression. In some embodiments, the expression level is normalized to GUSB. In some embodiments, the cancer overexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.

[0139] In some embodiments, the cancer is gastric cancer, which includes FGFR2 gene amplification. In some embodiments, the gastric cancer including FGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, the gastric cancer including FGFR2 amplification overexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In some embodiments, the gastric cancer including FGFR2 amplification expresses FGFR2IIIb at a normalization level greater than 2, 3, 5, or 10 times the normalization level of FGFR2IIIc expression. In some embodiments, the expression level is normalized to GUSB. In some embodiments, the gastric cancer overexpresses FGFR2IIIb but does not include FGFR2 gene amplification. In some embodiments, the overexpression is mRNA overexpression. In some embodiments, the overexpression is protein overexpression.

[0140] FGFR2IIIb gene amplification can be determined by any suitable method in the art, including but not limited to in situ hybridization (ISH). In some embodiments, FGFR2 amplification comprises more than 3 FGFR2:CEN10 (chromosome 10 centromeres).

[0141] FGFR2IIIb mRNA overexpression can be determined by any suitable method in the art, including but not limited to methods including quantitative PCR (qPCR). The term "FGFR2IIIb mRNA overexpression" means elevated levels of FGFR2IIIb mRNA regardless of the cause of the elevated levels (i.e., whether the elevated levels are the result of increased transcription and / or decreased degradation of mRNA, other mechanisms, or a combination of mechanisms).

[0142] FGFR2IIIb protein overexpression may be determined by any suitable method in the art, including but not limited to antibody-based methods, such as immunohistochemistry (IHC). In some embodiments, IHC staining is scored according to methods in the art. The term “FGFR2IIIb protein overexpression” means an elevated level of FGFR2IIIb protein regardless of the cause of the elevated level (i.e., whether the elevated level is the result of increased translation and / or decreased degradation of the protein, other mechanisms, or a combination of mechanisms). In some embodiments, 1+, 2+, or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. For example, overexpression may be determined by an IHC signal of 1+, 2+, or 3+ in at least 10% of tumor cells, e.g., at least 20%, 30%, 40%, or 50% of tumor cells. In some embodiments, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIb overexpression. For example, overexpression may be determined by an IHC signal of 2+ or 3+ in at least 10% of tumor cells, e.g., at least 20%, 30%, 40%, or 50% of tumor cells.

[0143] In some embodiments, FGFR2 overexpression may be recorded as an "H score." For example, in some such embodiments, the tumor is a bladder cancer tumor. To determine the H score, the first membrane staining intensity may be determined for cells in a fixed field to obtain a score of 0, 1+, 2+, or 3+ via IHC, and the H score may be calculated using the following formula: 1 × (%) of cells visualized with an IHC intensity of 1+ + 2 × (%) of cells visualized with an IHC intensity of 2+ + 3 × (%) of cells visualized with an IHC intensity of 3+. Theoretically, the H score may range from 0 to 300, and is 300 when all cells in the field have an IHC stain of 3+. In some embodiments, the patient to be treated has an initial H score for FGFR2, e.g., FGFR2b (e.g., FGFR2IIIb), greater than 20, e.g., greater than 30, greater than 40, greater than 50, or greater than 100, or in the range of 20 to 300, 20 to 100, 20 to 50, 20 to 40, or 20 to 30. In some embodiments, the patient has an H score greater than 10 or is within the range of 10 to 20 or 15 to 20. In other embodiments, the patient has an H score of 0 to 10, which may indicate a lack of FGFR2 overexpression. In some such embodiments, the patient is a bladder cancer patient.

[0144] In some embodiments where the patient has gastric cancer or bladder cancer, the subject may have been previously determined to have one of the following profiles, or alternatively, the treatment method comprises determining whether the patient is suitable for one of the following profiles for FGFR2 expression / gene amplification, wherein the following profiles may represent the level of predicted responsiveness to treatment: a) for gastric cancer subjects, an IHC signal of 3+ in at least 10% of tumor cells; b) for gastric cancer subjects, an IHC signal of 3+ in at least 10% of tumor cells as well as amplification of the FGFR2 gene; c) for gastric cancer subjects, an IHC signal of 3+ in at least 10% of tumor cells without amplification of the FGFR2 gene; d) for gastric cancer subjects, an IHC signal of 1+ or 2+ in at least 10% of tumor cells; e) for bladder cancer subjects, an IHC signal of 1+ in at least 10% of tumor cells; f) for bladder cancer subjects, an IHC signal of 2+ in at least 10% of tumor cells; g) for bladder cancer subjects, an H score greater than 20; h) for bladder cancer subjects, an H score between 10 and 19; i) for bladder cancer subjects, an H score less than 10.In some embodiments where the patient has gastric cancer or bladder cancer, the subject may have been previously determined to have one of the following profiles, or alternatively, the treatment method comprises determining whether the patient is suitable for one of the following profiles for FGFR2 expression / gene amplification, wherein the following profiles may represent the level of predicted responsiveness to treatment: a) for gastric cancer subjects, an IHC signal of 3+ in at least 5% of tumor cells; b) for gastric cancer subjects, an IHC signal of 3+ in at least 5% of tumor cells as well as amplification of the FGFR2 gene; c) for gastric cancer subjects, an IHC signal of 3+ in at least 5% of tumor cells without amplification of the FGFR2 gene; d) for gastric cancer subjects, an IHC signal of 1+ or 2+ in at least 5% of tumor cells; e) for bladder cancer subjects, an IHC signal of 1+ in at least 5% of tumor cells; f) for bladder cancer subjects, an IHC signal of 2+ in at least 5% of tumor cells; g) for bladder cancer subjects, an H score greater than 20; h) for bladder cancer subjects, an H score between 10 and 19; i) for bladder cancer subjects, an H score less than 10.In some embodiments where the patient has gastric cancer or bladder cancer, the subject may have been previously determined to have one of the following profiles, or alternatively, the treatment method comprises determining whether the patient is suitable for one of the following profiles for FGFR2 expression / gene amplification, wherein the following profiles may represent a level of predicted responsiveness to treatment: a) the cancer is gastric cancer with an FGFR2-IIIb immunohistochemistry (IHC) signal of 2+ or 3+ in a cancer sample; b) the cancer is gastric cancer with an FGFR2-IIIb IHC signal of 2+ or 3+ in a cancer sample and an amplified FGFR2 gene; c) the cancer is gastric cancer with an FGFR2-IIIb IHC signal of 2+ or 3+ in a cancer sample and an unamplified FGFR2 gene; or d) the cancer is bladder cancer with an FGFR2-IIIb IHC signal of 2+ or 3+ in a cancer sample.

[0145] In some embodiments, the anti-FGFR2 antibody may be administered to a cancer patient at a dose within a limited range of at least 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mg / kg, or any two of these doses. In some embodiments, the antibody formulation is administered once every 1, 2, 3, 4, or 5 weeks.

[0146] In some embodiments, a pharmaceutical formulation comprising an anti-FGFR2 antibody is provided as part of a combination of an agent, e.g., an immune checkpoint inhibitor (immunostimulator) and a chemotherapy agent. For example, the anti-FGFR2 antibody or a formulation comprising it may be provided before, substantially simultaneously with, or after another mode of treatment, e.g., surgery, chemotherapy, or radiotherapy. In some embodiments, an effective amount of the formulation of this specification comprising an anti-FGFR2 antibody is administered together with another anticancer agent. Non-limiting exemplary anticancer agents that may be administered with anti-FGFR2 antibodies include platinum agonists (e.g., cisplatin, oxaliplatin, and carboplatin), paclitaxel (TAXOL®), albumin-modified nanoformulations of paclitaxel (ABRAXANE®), docetaxel (TAXOTERE®), gemcitabine (GEMZAR®), capecitabine (XELODA®, irinotecan (CAMPTOSAR®), epirubicin (ELLENCE®, PHARMORUBICIN®), FOLFOX (oxaliplatin combined with 5-FU and leucovorin), FOLFIRI (combination of leucovorin, 5-FU, and irinotecan), leucovorin, and fluorouracil (5-FU, Includes EFUDEX®), mitomycin C (MITOZYTREX™, MUTAMYCIN®), and doxorubicin hydrochloride (Adriamycin PFS, Adriamycin RDF, RUBEX®). In some embodiments, an effective amount of a formulation containing an anti-FGFR2 antibody is administered with paclitaxel. In some embodiments, an effective amount of an anti-FGFR2 antibody formulation is administered with cisplatin and / or 5-FU. In some embodiments, an effective amount of an anti-FGFR2 antibody formulation is administered with FOLFOX (oxaliplatin, 5-FU, and leucovorin).

[0147] In some embodiments, a method for treating cancer is provided, comprising the step of administering an effective amount of a pharmaceutical formulation comprising an anti-FGFR2 antibody and at least one immunostimulator. In some other embodiments, a method for treating cancer is provided, comprising the step of administering an effective amount of a formulation comprising an anti-FGFR2 antibody and an effective amount of at least one immunostimulator. In exemplary embodiments, at least one immunostimulator comprises a PD-1 / PD-L1 inhibitor. In some embodiments, a formulation comprising an anti-FGFR2 antibody and at least one immunostimulator, e.g., a PD-1 / PD-L1 inhibitor, is administered simultaneously. In some embodiments, a formulation comprising an anti-FGFR2 antibody and at least one immunostimulator, e.g., a PD-1 / PD-L1 inhibitor, is administered sequentially. In some embodiments, at least one, at least two, at least three, at least five, or at least ten doses of the anti-FGFR2 antibody are administered prior to the administration of at least one immunostimulator, e.g., a PD-1 / PD-L1 inhibitor. In some embodiments, at least one immunostimulator, such as a PD-1 / PD-L1 inhibitor, is administered in at least one, at least two, at least three, at least five, or at least ten doses prior to administration of a formulation containing an anti-FGFR2 antibody. In some embodiments, the final dose of at least one immunostimulator, such as a PD-1 / PD-L1 inhibitor, is administered at least 1, 2, 3, 5, or 10 days prior to the first administration of the anti-FGFR2 antibody, or 1, 2, 3, 5, 12, or 24 weeks prior. In some other embodiments, the final dose of the anti-FGFR2 antibody is administered at least 1, 2, 3, 5, or 10 days prior to the first administration of at least one immunostimulator, such as a PD-1 / PD-L1 inhibitor, or 1, 2, 3, 5, 12, or 24 weeks prior.In some embodiments, the subject has received or is receiving PD-1 / PD-L1 inhibitor therapy, and an anti-FGFR2 antibody is added to the therapeutic regimen.

[0148] Examples

[0149] The embodiments discussed below are intended to be purely illustrative of the invention and should not be construed as limiting the invention in any way. While efforts have been made to ensure accuracy regarding the numbers used (e.g., quantities, temperatures, etc.), some experimental error and deviation must be taken into account.

[0150] Examples 1: Formulation Materials and methods used in the study

[0151] I. Antibody production

[0152] The anti-FGFR2-IIIb antibody used in the following examples was generated from Chinese hamster ovary (CHO) cell lines lacking the FUT8 gene (α1,6-fucosyltransferase), and various lots that underwent changes during the fermentation and purification processes were used for antibody development. The anti-FGFR2-IIIb antibody is a defucosylated humanized IgG1 monoclonal antibody. A knowledge-based formulation development approach was used to identify an appropriate composition that provides maximum stability for the protein. To this end, both the endogenous properties of the molecule and exogenous formulation components that may affect the stability of the protein were considered. Studies were conducted to identify appropriate components for producing a liquid formulation of 10 to 20 mg / ml that can be diluted for IV infusion.

[0153] Raw materials: L-histidine (PN H3911), L-histidine.HCl (PN H5659), and sodium chloride (PN S9888) were obtained from Sigma Aldrich. L-arginine.HCl (PN 2067-06), citric acid (PN 0119), sodium citrate (PN 3627), and polysorbate 20 (PN 4116) were obtained from JT Baker (Thermo Fischer Scientific).

[0154] Containers / Capsules: 3 cc Type I West Pharmaceutical glass vials (PN 68000368) and 13 mm caps (PN 19700116) from West Pharmaceutical Services, Inc. (West Pharmaceutical Services, Inc.; Pennsylvania, USA), polypropylene tubes, and glass HPLC vials were used to fill the formulation.

[0155] II. General formulation procedure

[0156] The formulation was prepared by dialyzing the drug substance with a target formulation buffer using an MWCO 20 kD dialysis membrane. The formulation was filtered in a laminar flow hood using a 0.2 µm filter unit and filled into an appropriate container / stopper system for stability evaluation. Individual vials were withdrawn at predetermined times under specified storage conditions for analysis.

[0157] III. Analysis method

[0158] Stability samples were analyzed using the initial development analysis described below.

[0159] Visual inspection Visual evaluation was performed on a black and white background under fluorescent lighting.

[0160] Protein concentrationProtein concentration was determined by UV absorbance at 280 nm using a theoretical absorption coefficient of 1.43 cm⁻¹ [g / ℓ]⁻¹. Samples were diluted within the linear absorbance range using appropriate formulation buffers and measured against Dulbecco phosphate-buffered saline (DPBS) water. OD measurements were performed on a Beckman Coulter DU800 UV-Vis spectrophotometer (Beckman Coulter, Incorporated, California, USA).

[0161] pH : The buffer pH was determined using a calibrated Beckman Coulter pHi560 pH meter (Beckman Coulter, Incorporated, California, USA).

[0162] osmotic pressure The buffer osmotic pressure was measured by vapor pressure using the Wescor VAPRO® system (Wescor, Incorporated, Utah, USA).

[0163] Differential Scanning Calorimetry ( DSC ) analyze High-sensitivity differential scanning calorimetry (HSDSC) analysis was performed using VP-DSC (MicroCal, Northampton, Massachusetts, USA). Approximately 0.5 mg of each sample was loaded into a sample cell, and an equal amount of matching dialysis buffer was loaded into a reference cell. Samples were scanned at a rate of 1°C / min at 20 to 90°C or 100°C. Data were analyzed using Origin 7.0 data analysis software (OriginLab, Massachusetts, USA).

[0164] ion exchange Chromatography (IEX)Cation exchange chromatography is a method for separating protein variants based on net surface charge. While the isoelectric point (pI, the pH at which the protein has no net charge) of a protein is determined by its primary amino acid sequence, the net charge of the protein variant is based on the protein's isoelectric charge and the pH of the performance buffer. For example, in a performance buffer at pH 7, which is about 1 unit below the antibody's pI of 8.4, the protein has a net positive charge and electrostatically binds to an oppositely weakly charged acidic carboxylate functional group located on the ethylvinylbenzene-divinylbenzene substrate and the cross-linked Propac WCX-10 column resin. Using a high-performance liquid chromatography system equipped with UV detection at 280 nm, protein variants are separated and eluted from the column when an increasing salt gradient is applied, where variants with the weakest ionic interactions begin to elute first, and variants with stronger ionic interactions elute later at higher salt concentrations. The isoproteins eluted sequentially into three regions: acidic, major, and basic, with the highest peak being the major isoprotein peak. Based on the peak regions, each region had a calculated relative peak percentage. A weak cation exchange HPLC method was performed using a Dionex WCX-10 PROPAC™ 4×250 mm column (Thermo Fisher Scientific). Samples were run on an Agilent 1200 Series HPLC, and chromatograms were integrated using Chemstation software (Agilent Technologies, California, USA). Mobile phase A was 10 mM HEPES at pH 7.0, and mobile phase B was 10 mM HEPES at pH 7.0 and 100 mM sodium chloride. A gradient method was used at 40 to 90% B for 28 minutes, and UV detection was performed at 280 nm. Acidity, basicity, and major peak percentages were recorded from the total area percentage of each peak-associated absorbance.

[0165] Size-Exclusion Chromatography (SEC) Size exclusion chromatography is a method for separating proteins based on their size. This method uses porous resins to separate molecules of different sizes. Larger molecules elute earlier because they cannot access the resin pores and pass through the column without obstruction. Smaller molecules can access the resin pores, and the tortuosity within the resin particles can increase the path length through the column. Therefore, smaller molecules elute after larger molecules. Two methods were used for size exclusion chromatography. For the first method, a Tosoh G3000SWXL 7.8×300 mm column was used with 100 mM sodium phosphate and 700 mM arginine at pH 6.8 as the mobile phase for 30 minutes at 0.5 mL / min (Sigma-Aldrich, Incorporated, Missouri, USA). The second method utilized a Sepax Zenix SEC-300 7.8×200 mm column with 100 mM sodium and 400 mM sodium chloride at pH 6.8 as the mobile phase for 12 minutes at 1 mL / min (Sepax Technologies, Incorporated, Delaware, USA). Samples were run on Agilent 1100 and 1200 series HPLCs, and chromatograms were integrated using ChemStation software with detection set at 280 nm. The percentage of aggregates, low molecular weight (LMW), and major peaks were recorded as the percentage of the total area of ​​the absorbance associated with each peak.

[0166] light scattering : Ultraviolet absorbance at 280 nm using a spectrophotometer (UV A 280 ) was measured. Beer's law, that is, A = ε l cThe concentration of the antibody samples was calculated using (ε = absorption coefficient; l = length of the solution through which light passes (path length of the cuvette) (cm), and c = concentration of the solution). The theoretical absorption coefficient was used to determine the antibody concentration (e.g., the theoretical absorption coefficient for the anti-FGFR2 antibody is 1.43 (OD·mL) (mg·cm)). The concentration (unit: mg / mL) for each sample preparation was calculated as follows: Concentration = (Absorbance at A280 - Absorbance at A350) × (Dilution factor) / (Absorbance coefficient × Path length). The mean and standard deviation were calculated for each sample.

[0167] Examples 2: Solubility in water

[0168] The anti-FGFR2-IIIb antibody was concentrated to approximately 100 mg / mL and dialyzed in water. The sample was then further concentrated to 180 mg / mL. The anti-FGFR2-IIIb antibody was soluble in water at 180 mg / mL. It did not reach maximum solubility.

[0169] Examples 3: Hang- FGFR2 - IIIb Initial freeze / thaw stability of antibodies

[0170] A freeze-thaw study was performed on the anti-FGFR2-IIIb antibody. The antibody was concentrated to 11.6 mg / mL and formulated in 1× PBS at pH 7.4, with or without 0.01% polysorbate 20. The formulated solution was filled into vials, and freeze-thaw cycles were performed up to three times at -70°C to ambient temperatures. Sample vials were analyzed by visual inspection and SE-HPLC. The freeze-thaw results are shown in Table 1. A slight increase in aggregates was observed over the three freeze-thaw cycles. The results suggest that 1× PBS is acceptable for the formulation.

[0171]

[0172] Examples 4: Hang- FGFR2 - IIIb Antibody oxidation research

[0173] The anti-FGFR2-IIIb antibody has a total of 10 methionine residues; four of the methionines are in each heavy chain, and none of the methionines are located on CDR1. Additionally, there is one methionine in the light chain.

[0174] Experiments were conducted to evaluate the sensitivity of methionine to oxidation by incubating anti-FGFR2-IIIb antibodies with 0.01%, 0.1%, or 1.0% hydrogen peroxide overnight at room temperature. Subsequently, the oxidized samples were characterized by trypsin mapping LC / MS to qualitatively determine the oxidation levels of each methionine residue. The activity of the oxidized samples was determined by cell-based assays for efficacy, and binding affinity was analyzed using Viacore™ (GE Healthcare). The peptide mapping LC / MS characterizations shown in Table 2 revealed that light-chain methionine 5 and medium-chain methionine 81 were less sensitive to oxidation. Medium-chain methionine 249, 355, and 425 were more sensitive to oxidation. All oxidized samples exhibited efficacy similar to the control group by ELISA analysis.

[0175]

[0176] The binding of the anti-FGFR2-IIIb antibody to FGFR2b and protein A was measured by Viacore. Oxidation of heavy chain methionine did not interfere with the binding of the anti-FGFR2-IIIb antibody to FGFR2b (Figure 1 and Table 3). Increased oxidation interfered with binding to protein A. Considering that the activity of the anti-FGFR2-IIIb antibody was not significantly affected in highly oxidized samples, the oxidative degradation of the anti-FGFR2-IIIb antibody was not significant.

[0177]

[0178] Examples 5: Protein Regarding stability buffer The effect of pH

[0179] Formulation pH affects protein stability. The pH of the formulation can influence biochemical degradation pathways, e.g., deamidation, isomerization, and oxidation, as well as biophysical degradation, e.g., aggregation and fragmentation resulting from interactions between proteins and their environment. Buffer pH was investigated as a formulation variable that may affect product stability. The effect of pH on the stereochemical stability and structural stability of the anti-FGFR2-IIIb antibody during storage was evaluated. The anti-FGFR2-IIIb antibody was purified in two steps and formulated into nine different isotonic formulations (Formulations 1 to 9 in Table 4) with pH values ​​ranging from 4.0 to 8.0. The protein concentration of the buffer exchange solution was adjusted to 1 mg / mL, and the solution was filled into vials for DSC analysis and evaluation of isothermal stability at 40°C and 25°C. The evaluated formulations are listed in Table 4.

[0180]

[0181] Differential Scanning Calorimetry ( DSC ) Structural stability based on analysis

[0182] DSC thermograms were generated and analyzed for formulations 1 to 9 in Table 4. Formulations 1 to 3 are marked with * in Table 5, and the results are shown in Figure 2. Three transitions were observed upon heating of the samples. After the release transition, IgG molecules formed insoluble aggregates and precipitated from the solution. Table 5 shows the release temperature (T M 1) increases with increasing pH and reaches a maximum T at approximately 69°C at pH 6 and pH 7. M It indicates that it reached 1. The buffer species did not have a significant effect on the annealing temperature.

[0183]

[0184] Isothermal stability

[0185] The effect of buffer pH on the formation of aggregates, clips (fragments), and charged variants was evaluated by monitoring sample stability under accelerated storage conditions at 40°C and 25°C.

[0186] Changes in aggregates and clips were determined by SE-HPLC. Representative SE-HPLC chromatograms of selected anti-FGFR2-IIIb antibody pH screening samples are shown in Figure 3. The main peak represents the monomer, the peak eluted before the monomer is the aggregate, and the peak eluted later is the clip. Soluble aggregates increased rapidly in the pH 4 formulation at 40°C; moderate increases in soluble aggregates with increasing pH were observed in pH 5.0 and pH 8.0 buffers. A similar effect of pH on clip formation was observed.

[0187] The effect of buffer pH on aggregates (Fig. 4) and clips (Fig. 5) at 40°C was analyzed by SE-HPLC. Formulations 1, 2, 3, 7, 8, and 9 listed in Table 5 were tested. Within the tested pH range, clip formation was most pronounced at pH 4. At neutral pH, clip formation was negligible even after storage for one month at 40°C, as shown in Fig. 5. No apparent increase in aggregates was observed in the formulations from pH 5 to pH 8 after storage for at least two months at 25°C (Fig. 6). No apparent increase in clips was observed in the formulations from pH 5 to pH 8 after storage for at least two months at 25°C (Fig. 7).

[0188] Changes in the distribution of charged variants are another common degradation pathway for antibodies and are typically associated with the pH of the formulation buffer. Charged variants of the anti-FGFR2-IIIb antibody were analyzed using the weak cation-exchange HPLC (WCX-HPLC) method. A representative chromatogram is shown in Fig. 8. Peaks appearing before the main peak represent acidic species, and peaks appearing after the main peak represent basic species. The increase in acidic species is attributed to the deamidation of asparagine in IgG molecules. Changes in the charged variant profile occurred rapidly at 40°C. Therefore, changes in the charge profile were monitored at lower temperatures, e.g., 25°C. Figs. 9 through 11 illustrate the effect of buffer pH on charged variants at 25°C as determined by weak cation-exchange high-performance liquid chromatography (WCX-HPLC). As shown in Fig. 9, acidic species increased rapidly under basic pH conditions, particularly at pH 8.0. Samples formulated at pH 5 to pH 6 remained stable even after 2 months of storage at 25°C. Figure 10 shows the effect of pH on the percentage of basic variants during storage at 25°C for 0 to 2.5 months. Figure 11 shows the effect of pH on the main peak during storage at 25°C for 0 to 2.5 months.

[0189] The results of the pH screening study indicate that the stability of the anti-FGFR2-IIIb antibody under stress storage conditions depends significantly on the pH of the formulation. At basic pH, the aggregation and formation of acidic variants were the major degradation pathways. The anti-FGFR2-IIIb antibody was determined to be most stable in the pH range of 5.0 to 6.0.

[0190] Examples 6: Protein Effect of excipients on stability

[0191] Formulation excipients, such as buffer types and extenders, were analyzed for their effect on product stability. To evaluate the effect of excipients on the stability of the anti-FGFR2-IIIb antibody, various citrate, phosphate, and histidine buffers were tested. Seven different isotonic solutions buffered at pH 6.0 tested are listed in Table 6.

[0192]

[0193] The protein concentration was adjusted to 20 mg / mL, and the solution was filled into vials. Formulations 1 to 4 in Table 6 were analyzed by DSC. Physical stability was evaluated for all formulations, and isothermal stability was evaluated for formulations 1 to 5 at 40°C, 25°C, and 5°C.

[0194] DSC Structural stability based on analysis

[0195] The unfolding temperatures of various formulations of the anti-FGFR2-IIIb antibody were determined by DSC analysis and are summarized in Table 7. Precipitation was observed in all formulations after heating to 90°C. In the formulations, the surfactant did not prevent the antibody from precipitating after denaturation. As shown in Table 7, the formulations had similar melting temperatures.

[0196]

[0197] Three thermal transitions were detected when heating various formulations of the anti-FGFR2-IIIb antibody. The anti-FGFR2-IIIb antibody began to unwind at 52.7°C in the pH 4 formulation, and the unwinding temperature (Tm) increased with increasing pH, reaching a maximum Tm1 at approximately 70°C at pH 6 and pH 7, and Tm decreased slightly at pH 8.

[0198] Physical stability

[0199] Since protein pharmaceuticals are prone to aggregation when exposed to shear stress, physical stability studies were conducted to evaluate the effect of excipients on the stability of anti-FGFR2-IIIb antibodies against multiple freeze / thaw cycles and vigorous stirring. Stability was assessed by visual observation of insoluble and soluble aggregates, A350 light scattering, and SE-HPLC. The antibody concentration in the samples was 20 mg / mL.

[0200] The sample vials were placed horizontally on an orbital shaker and subjected to stirring stress by shaking the samples at 500 RPM at room temperature for 77 hours. To evaluate the effect of polysorbate 20 on preventing aggregation formation, a head-to-head comparison was performed on arginine formulations containing 0%, 0.01%, 0.05%, and 0.10% polysorbate 20. As shown in Table 8 and Figure 12, no apparent increase in soluble aggregates was detected by SE-HPLC in any of the formulations.

[0201] A freeze / thaw study was also performed by freezing the samples at -70°C and thawing them at ambient temperature over five cycles. As shown in Figure 13, the only apparent change was observed in soluble aggregates in the histidine / NaCl formulation, which increased with increasing freeze / thaw cycles. The addition of 0.01% polysorbate 20 to the formulation improved the stability of the anti-FGFR2-IIIb antibody against shaking stress.

[0202]

[0203] Isothermal stability

[0204] The effects of buffer species and extenders on the formation of aggregates, clips, and charged variants were evaluated by monitoring the stability of the anti-FGFR2-IIIb antibody under accelerated storage conditions at 40°C, 25°C, and 5°C. Changes in aggregates and clips were monitored using SE-HPLC. The following formulations were tested: 20 mM citrate, 150 mM L-arginine, and 0.01% polysorbate 20; 20 mM phosphate, 150 mM L-arginine, and 0.01% polysorbate 20; 20 mM histidine, 150 mM L-arginine, and 0.01% polysorbate 20; 20 mM histidine, 150 mM NaCl, and 0.01% polysorbate 20; and 20 mM histidine, 150 mM L-arginine, polysorbate 20 none.

[0205] As shown in Fig. 14, aggregates formed at a higher rate in the citrate and phosphate formulations at 40°C. Minor fragmentation was observed in all formulations after 1 month of storage at 40°C. All formulations showed the same increase in aggregation after 3 months of storage at 25°C (Fig. 15).

[0206] The formulation excipients did not affect the charge profile of the anti-FGFR2-IIIb antibody during storage. As shown in Figure 16, there was an increase in acidic variants in all formulations. Figure 17 shows that there was a decrease in basic variants over time in all formulations, and Figure 18 shows the effect of excipients on the major peaks over time.

[0207] Based on antibody stability studies, L-histidine did not have a significant effect on antibody stability among the buffer species evaluated. In sodium citrate and sodium phosphate, aggregates increased after 1 month at 40°C. Therefore, L-histidine is more stable than sodium citrate or sodium phosphate; aggregates increased at a faster rate in the citrate and phosphate formulations.

[0208] Among the evaluators, NaCl produced aggregate formation induced by vigorous shaking and exposure to freezing / thawing processes. The most stable antibody profile was obtained with the L-arginine formulation.

[0209] Examples 7: pH Evaluation Study

[0210] The effect of pH on the chemical and physical stability of the formulations was tested. The following formulations were tested at pH 5.5, 6.0, and 6.5: (a) 20 mM histidine, 150 mM arginine, 0.01% polysorbate 20 and (b) 20 mM histidine, 270 mM sucrose, 0.01% polysorbate 20 (Table 9). The formulations were adjusted to 20 mg / mL and filled into glass vials.

[0211]

[0212] Effect of pH on physical stability

[0213] Stability against multiple freezing / freezing cycles and vigorous stirring was evaluated. Stability was assessed by visual observation of insoluble and soluble aggregates, A350 light scattering, and SE-HPLC.

[0214] I. mechanical stirring

[0215] Stirring stress was applied to the samples by placing the sample vials horizontally on an orbital shaker and shaking at 500 RPM at room temperature for 72 hours. No clear evidence of soluble aggregates was detected by SE-HPLC in any of the formulations. Figure 19 shows the effect of mechanical stress on aggregation in the histidine / arginine and histidine / sucrose formulations over time at pH 5.5 to 6.5 (pH 5.5, 6.0, and 6.5). Figure 19 shows the effect of mechanical stress on the clips in the histidine / arginine and histidine / sucrose formulations at pH 5.5 to 6.5. After 72 hours of mechanical stirring, there was no significant change in light scattering or concentration at A350 nm (as shown in Table 10).

[0216]

[0217] II. Freeze thawing stability

[0218] Freeze / thaw studies were performed by freezing samples at -70°C and thawing them at ambient temperature over five cycles. Figures 21 and 22 show the effects of freeze-thaw on aggregation and clipping in histidine / arginine and histidine / sucrose formulations at pH 5.5 to 6.5. In Figure 21, there was no apparent increase in soluble aggregates detected by SE-HPLC in either the histidine / arginine or histidine / sucrose formulations at pH 5.5 to 6.5. In Figure 22, no apparent evidence of clipping was detected by SE-HPLC in any of the formulations.

[0219]

[0220] After freezing and thawing, the concentration and light scattering remained unchanged.

[0221] I. Effect of pH on isothermal stability

[0222] The effect of buffer pH on the formation of aggregates, clips, and charged variants was evaluated by monitoring sample stability under accelerated storage conditions at 40°C and 25°C, and real-time stability at 5°C. The following formulations were tested: (A) 20 mM histidine, 150 mM arginine, 0.01% polysorbate 20 ("histidine / arginine formulation"), and (B) 20 mM histidine, 150 mM sucrose, 0.01% polysorbate 20 ("histidine / sucrose formulation").

[0223] Aggregation and clip formation increased with pH in the histidine / arginine and histidine / sucrose formulations after 1 month at 40°C and 3 months at 25°C. The histidine / sucrose formulation showed a slightly lower aggregation rate. After 6 months at 5°C, aggregation remained unchanged, and clip formation increased by about 1% in both the histidine / arginine and histidine / sucrose formulations.

[0224] For example, FIGS. 23A to 23C show the formation of aggregates in the histidine / arginine formulation under various pH conditions measured over (A) 1 month at 40°C, (B) 3 months at 25°C, and (C) 6 months at 5°C. FIGS. 24A to 24C show the formation of clips in the histidine / arginine formulation under various pH conditions (pH 5.5 to 6.5) measured over (A) 1 month at 40°C, (B) 3 months at 25°C, and (C) 6 months at 5°C.

[0225] FIGS. 25A to 25C show aggregate formation in the histidine / sucrose formulation under various pH conditions measured over (A) 1 month at 40°C, (B) 3 months at 25°C, and (C) 6 months at 5°C. FIGS. 26A to 26C show antibody clip formation in the histidine / sucrose formulation under various pH conditions (pH 5.5 to 6.5) measured over (A) 1 month at 40°C, (B) 3 months at 25°C, and (C) 6 months at 5°C.

[0226] The charged isoform also remained unchanged after 6 months at 5°C. For example, FIGS. 27A to 27C show acidic variants in the histidine / sucrose formulation with increased acidity after (A) 1 month at 40°C, (B) 3 months at 25°C, and (C) 6 months at 5°C.

[0227] Additionally, FIGS. 28A to 28C illustrate the effect of pH between 5.5 and 6.5 on the formation of acidic variants in a histidine / arginine formulation comprising 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. FIG. 28A illustrates the formation of acidic variants, indicating that the % of acidic variants generally remained below 40% after 1 month at 40°C at pH 5.5 to 6.0. FIG. 28B illustrates the formation of acidic variants, indicating that the % of acidic variants generally remained below 30.0% after 3 months at 25°C at pH 5.5 to 6.0. FIG. 28C illustrates the formation of acidic variants, indicating that the % of acidic variants generally remained below 25% after 6 months at 5°C at all pH values. FIGS. 29A to 29C show the effect of pH between 5.5 and 6.5 on the formation of basic variants in a histidine / sucrose formulation containing 20 mM histidine, 270 mM sucrose, and 0.01% polysorbate 20. FIG. 29A shows the formation of basic variants, indicating that the percentage of basic variants mostly remained between 10% and 20% after 1 month at 40°C at pH between 5.5 and 6.0. FIG. 29B shows the formation of basic variants, indicating that the percentage of basic variants generally remained between 20% and 30% after 3 months at 25°C at pH between 5.5 and 6.0. Figure 29C shows the formation of basic variants, indicating that the percentage of basic variants was generally maintained at 25% to 30% after 6 months at 5°C at all pH levels.

[0228] FIGS. 30A to 30C show the effect of a pH between 5.5 and 6.5 on the formation of basic variants in a histidine / arginine formulation containing 20 mM histidine, 150 mM arginine, and 0.01% polysorbate 20. FIG. 30A shows the formation of basic variants, indicating that the percentage of basic variants remained mostly between 10% and 20% after 1 month at 40°C at a pH between 5.5 and 6.0. FIG. 30B shows the formation of basic variants, indicating that the percentage of basic variants generally remained between 15% and 25% after 3 months at 25°C at a pH between 5.5 and 6.0. Figure 30C shows the formation of basic variants, indicating that the percentage of basic variants was generally maintained at 25% to 30% after 6 months at 5°C at all pH levels.

[0229] Both the histidine / arginine and histidine / sucrose formulations maintained acceptable stability at pH 5.5 to 6.5. Overall, the histidine / sucrose formulation was more stable than histidine / arginine at pH 5.5 to 6.0.

[0230] Based on data regarding formulation stability at 25°C for 3 months and stability at 5°C for 6 months, and data from other formulation studies of the above examples, a liquid formulation was developed comprising 20 mg / ml anti-FGFR2-IIIb antibody, 20 mM L-histidine, 270 mM sucrose, and 0.01% polysorbate 20 at pH 6.0. Additionally, a second formulation was developed comprising 20 mg / ml anti-FGFR2-IIIb antibody, 20 mM L-histidine, 150 mM L-arginine, and 0.01% polysorbate 20 at pH 5.7.

[0231] The scope of the present invention should not be limited by the specific embodiments described herein. In fact, various variations provided herein in addition to those described will be apparent to those skilled in the art from the description and the accompanying drawings. Such variations are intended to fall within the scope of the appended claims.

[0232] All references cited in this specification (e.g., publications or patents or patent applications) are incorporated by reference into this specification for all purposes to the same extent that each individual reference (e.g., publications or patents or patent applications) is specifically and individually indicated as being included by reference in its entirety for all purposes.

[0233] Other embodiments fall within the scope of the following claims.

[0234] Ranking table

[0235] The following sequence table provides specific sequences discussed herein. All polypeptide and antibody sequences are shown without leader sequences unless otherwise indicated.

[0236] Sequence and Description Table

[0237]

[0238]

[0239]

Claims

Claim 1 A pharmaceutical formulation comprising: i) 20 mg / ml of anti-FGFR2 antibody, comprising the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence of SEQ ID NO. 3 and defucosylated anti-FGFR2 antibody; ii) 20 mM L-histidine buffer; iii) 270 mM sucrose; and iv) 0.01% polysorbate 20, wherein the formulation is a non-lyophilized liquid pharmaceutical formulation, and the formulation has a pH of 6.

0. Claim 2 In claim 1, a pharmaceutical formulation having one or more of the following characteristics: (a) an increase in protein aggregation in the formulation of 2.0% or less after storage at 5°C for 6 months; (b) an increase in protein aggregation in the formulation of 2.0% or 2.5% or less after storage at 25°C for 3 months; (c) an increase in protein aggregation in the formulation of 7.0% or less after storage at 40°C for 3 months; (d) no change in charged protein variants in the formulation of more than 5% after storage at 5°C for 6 months; (e) an increase in protein aggregation in the formulation of 2.0% or less after 5 freeze-thaw cycles at -70°C; (f) an increase in protein aggregation in the formulation of 2.0% or less after 72 hours of mechanical stress at 500 rpm; (g) dilution with a saline solution before intravenous administration; (h) administration by intravenous administration; and / or (i) isotonic with human plasma. Claim 3 A pharmaceutical formulation contained in a single-use vial in claim 1. Claim 4 A pharmaceutical formulation for use in treating a solid tumor in a patient requiring treatment for a solid tumor, according to claim 1. Claim 5 A pharmaceutical formulation according to paragraph 4, wherein the above use involves intravenous administration to a patient. Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete

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