Pharmaceutical formulations of fusion proteins

Abstract

Provided herein is a liquid pharmaceutical composition comprising a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to human epidermal growth factor receptor (hEGFR) and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of human transforming growth factor beta receptor II (hTGFβRII), a buffer present at a concentration of about 5 mM to about 30 mM, and a tonicity adjusting agent present at a concentration of about 4% to about 10% w / v, and having a pH of about 5.5 to about 7.0. Methods for preparing liquid pharmaceutical compositions comprising the fusion protein and methods for use in treating cancer are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to Indian Provisional Application No. IN202011054539, filed on 15 December 2020, the full disclosure of which is incorporated herein by reference. [Background technology]

[0002] Therapeutic antibodies are large and complex molecules, and therefore subject to degradation processes, especially in liquid form. Multifunctional fusion proteins, particularly antibody fusion proteins, are even more complex, containing multiple functional domains of different structures and functions, either directly linked or linked via linkers. The instability in antibodies and fusion proteins makes the development of stable formulations suitable for delivery to target difficult. For example, these protein preparations may have short shelf lives, and proteins may lose their biological activity due to chemical and physical degradation during storage, especially long-term storage. Chemical degradation processes include, for example, deamidation, racemization, hydrolysis, oxidation, beta-elimination, and disulfide exchange. Physical degradation processes include, for example, denaturation, aggregation, sedimentation, and adsorption. Despite general structural similarities, the development of formulations of different monoclonal antibodies has not been simple, due to their inherent and unpredictable behavior in liquid formulations. This unpredictability is even greater for multifunctional fusion proteins (e.g., fusion proteins containing antibodies and other protein components), due to the relative importance of, for example, the primary sequence, structure, linkage, multifunctionality, and degradation pathways (e.g., denaturation, aggregation, surface adsorption, deamidation, oxidation, isomerization, fragmentation, etc.). Therefore, there remains a need for novel, stable formulations of multifunctional fusion proteins (e.g., those described herein) that, even after long-term storage, exhibit stability, show low or undetectable levels of physical or chemical degradation, and show little or no loss of multifunctional biological activity. [Overview of the project]

[0003] The present invention addresses the above need by providing a stable liquid pharmaceutical formulation of a multifunctional fusion protein (e.g., BCA101) as further described below. Specifically, the present invention provides a stable liquid pharmaceutical formulation of a bifunctional fusion protein disclosed herein. The formulation of the present invention is useful for administration to mammals, particularly to humans with cancer (e.g., by intravenous administration).

[0004] Accordingly, in one embodiment, the present disclosure provides a liquid pharmaceutical composition comprising: (a) a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety comprises a polypeptide that specifically binds to a membrane-bound target protein and has a basic isoelectric point (pI), and (ii) the immunomodulatory moiety comprises a polypeptide that specifically binds to a soluble target protein having an acidic pI, wherein the membrane-bound target protein and the soluble target protein are different; (b) a buffer present at a concentration of about 5 mM to about 30 mM; and (c) a tonicity modifier present at a concentration of about 4 wt / vol% to about 10 wt / vol%; wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 7.0.

[0005] Accordingly, in one embodiment, the present disclosure provides a liquid pharmaceutical composition comprising: (a) a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to human epidermal growth factor receptor (hEGFR), and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of human transforming growth factor-beta receptor II (hTGFβRII); (b) a buffer present at a concentration of about 5 mM to about 30 mM; and (c) a tonicity regulator present at a concentration of about 4 wt / vol% to about 10 wt / vol%; wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 7.0.

[0006] In some embodiments, the buffer is citrate phosphate buffer, citrate buffer, succinate buffer, or histidine buffer. In some embodiments, the buffer is citrate phosphate buffer. In some embodiments, the buffer is present at concentrations of about 5 mM to about 25 mM, 5 mM to about 20 mM, 5 mM to about 15 mM, 5 mM to about 10 mM, or 10 mM to about 30 mM. In some embodiments, the buffer is present at concentrations of about 5 mM to about 15 mM. In some embodiments, the buffer is present at concentrations of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM. In some embodiments, the buffer is present at a concentration of about 10 mM. In some embodiments, the buffer is about 10 mM citrate phosphate.

[0007] In some embodiments, the tension regulator is a sugar. In some embodiments, the tension regulator is a disaccharide. In some embodiments, the tension regulator is sucrose or trehalose. In some embodiments, the tension regulator is sucrose. In some embodiments, the tension regulator is present at concentrations of about 5 wt / vol% to about 10 wt / vol%, 6 wt / vol% to about 10 wt / vol%, 7 wt / vol% to about 10 wt / vol%, 8 wt / vol% to about 10 wt / vol%, 5 wt / vol% to about 9 wt / vol%, 5 wt / vol% to about 8 wt / vol%, 6 wt / vol% to about 9 wt / vol%, 6 wt / vol% to about 8 wt / vol%, 7 wt / vol% to about 9 wt / vol%, or 7 wt / vol% to about 8 wt / vol%. In some embodiments, the tension regulator is present at concentrations of about 5 wt / vol% to about 8 wt / vol%. In some embodiments, the tension modifier is present at concentrations of about 5% by weight / vol, 6% by weight / vol, 7% by weight / vol, 8% by weight / vol, 9% by weight / vol, or 10% by weight / vol. In some embodiments, the tension modifier is present at a concentration of about 8% by weight / vol. In some embodiments, the tension modifier is sucrose and is present at a concentration of about 8% by weight / vol.

[0008] In some embodiments, the liquid pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the surfactant comprises polysorbate 20. In some embodiments, the surfactant is present at a concentration of about 0.005 to 0.1% by weight / volume. In some embodiments, the surfactant is present in amounts of approximately 0.01-0.1 wt / vol%, 0.02-0.1 wt / vol%, 0.01-0.9 wt / vol%, 0.01-0.8 wt / vol%, 0.01-0.7 wt / vol%, 0.01-0.6 wt / vol%, 0.01-0.5 wt / vol%, 0.01-0.4 wt / vol%, 0.01-0.3 wt / vol%, 0.01-0.2 wt / vol%, 0.01-0.1 wt / vol%, 0.02-0.9 wt / vol%, 0.02-0.8 wt / vol%, 0.02-0.7 wt / vol%, 0.02- It exists at concentrations of 0.6 wt / vol.%, 0.02-0.5 wt / vol.%, 0.02-0.4 wt / vol.%, 0.02-0.3 wt / vol.%, 0.02-0.2 wt / vol.%, 0.02-0.1 wt / vol.%, 0.005-0.9 wt / vol.%, 0.005-0.8 wt / vol.%, 0.005-0.7 wt / vol.%, 0.005-0.6 wt / vol.%, 0.005-0.5 wt / vol.%, 0.005-0.4 wt / vol.%, 0.005-0.3 wt / vol.%, 0.005-0.2 wt / vol.%, or 0.005-0.1 wt / vol. In some embodiments, the surfactant is present at concentrations of about 0.01% by weight / volume, 0.02% by weight / volume, 0.03% by weight / volume, 0.04% by weight / volume, 0.05% by weight / volume, 0.06% by weight / volume, 0.07% by weight / volume, 0.08% by weight / volume, 0.09% by weight / volume, or 0.1% by weight / volume. In some embodiments, the surfactant is present at a concentration of about 0.02% by weight / volume. In some embodiments, the surfactant is polysorbate 20, present at a concentration of about 0.02% by weight / volume.

[0009] In some embodiments, the liquid pharmaceutical composition has a pH of about 5.5 to about 7.0, 6.0 to about 7.0, 5.5 to about 6.5, 5.5 to about 6.0, or 6.0 to about 6.5. In some embodiments, the liquid pharmaceutical composition has a pH of about 6.0 to about 6.5. In some embodiments, the liquid pharmaceutical composition has a pH of about 5.5, 6.0, 6.5, or 7.0. In some embodiments, the liquid pharmaceutical composition has a pH of about 6.0.

[0010] In some embodiments, the liquid pharmaceutical composition has a weight osmolality of about 150 mOsmol / kg to about 400 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has a weight osmolality of about 150 mOsmol / kg to about 350 mOsmol / kg, 150 mOsmol / kg to about 300 mOsmol / kg, 200 mOsmol / kg to about 400 mOsmol / kg, 250 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 350 mOsmol / kg, 250 mOsmol / kg to about 350 mOsmol / kg, or 250 mOsmol / kg to about 300 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has a weight osmolality of about 250 mOsmol / kg to about 350 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has a weight osmolality of about 250 mOsmol / kg, 300 mOsmol / kg, or 300 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has a weight osmolality of about 300 mOsmol / kg.

[0011] In some embodiments, the liquid pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at -20°C. In some embodiments, the liquid pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at 2-8°C.

[0012] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at -80°C. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at -20°C. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at 2-8°C.

[0013] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -80°C for 12 months, 18 months, or 24 months. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -20°C for 12 months, 18 months, or 24 months. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at 2-8°C for 12 months, 18 months, or 24 months.

[0014] In some embodiments, the liquid pharmaceutical composition is stable after one, two, three, four, or five freeze-thaw cycles.

[0015] In some embodiments, the fusion protein retains bifunctional activity, as measured by a bifunctional enzyme-linked immunosorbent assay (ELISA), for at least 12, 18, or 24 months when stored at -20°C. In some embodiments, the fusion protein retains bifunctional activity, as measured by a bifunctional enzyme-linked immunosorbent assay (ELISA), for at least 12, 18, or 24 months when stored at -20°C. In some embodiments, the fusion protein retains bifunctional activity, as measured by a bifunctional enzyme-linked immunosorbent assay (ELISA), for at least 12, 18, or 24 months when stored at 2–8°C.

[0016] In some embodiments, the liquid pharmaceutical composition contains the fusion protein in aggregate form in an amount of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0017] In some embodiments, the liquid pharmaceutical composition, after storage at -20°C or 2-8°C for 12, 18, or 24 months, has at least one property selected from the group consisting of (a) increased shelf life, (b) increased temperature stability, (c) decreased aggregate formation, (d) increased chemical stability, (e) decreased fragment formation, and / or (f) decreased viscosity, compared to a reference formulation.

[0018] In some embodiments, the liquid pharmaceutical composition, after storage at -20°C or 2–8°C for 12, 18, or 24 months, has at least one property selected from the group consisting of (a) a decrease in aggregate percentage as measured by size exclusion chromatography (SEC), (b) a higher monomer percentage as measured by SEC, and / or (c) a lower turbidity value in turbidimetric units (NTU), compared to a reference formulation.

[0019] In some embodiments, the fusion protein is present at concentrations of approximately 5–50 mg / ml, 5–40 mg / ml, 5–30 mg / ml, 5–25 mg / ml, 10–50 mg / ml, 20–50 mg / ml, 25–50 mg / ml, 20–50 mg / ml, 20–40 mg / ml, 20–30 mg / ml, 25–50 mg / ml, 25–40 mg / ml, or 25–30 mg / ml. In some embodiments, the fusion protein is present at concentrations of approximately 20–30 mg / ml. In some embodiments, the fusion protein is present at concentrations of approximately 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In some embodiments, the fusion protein is present at concentrations of approximately 25 mg / ml.

[0020] In some embodiments, the targeted portion that specifically binds to hEGFR comprises an antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof. In some embodiments, the antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof, is a full-length antibody, a single-strand variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v).

[0021] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR comprises VH, which includes VH CDR1, VH CDR2, and VH CDR3, wherein (a) VH CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1; (b) VH CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2; and (c) VH CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

[0022] In some embodiments, the antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof, is a VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein (a) VL CDR1 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, (b) VL CDR2 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, and (c) VL CDR3 comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0023] In some embodiments, the antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof, comprises a VH comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0024] In some embodiments, the antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof, comprises a VL comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0025] In some embodiments, the antibody that specifically binds to hEGFR, or a functional fragment or functional variant thereof, comprises a heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0026] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR consists of a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

[0027] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR includes a heavy chain consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9.

[0028] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR consists of a heavy chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

[0029] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR includes a light chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0030] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR consists of a light chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0031] In some embodiments, the antibody, or its functional fragment or functional variant, that specifically binds to hEGFR includes cetuximab, panitumumab, or a functional fragment or functional variant of either of the aforementioned.

[0032] In some embodiments, the immunomodulatory portion comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0033] In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion. In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker.

[0034] In some embodiments, the immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker of sufficient length so that the immunomodulatory portion and the targeting portion can bind to their respective targets simultaneously. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 24, 25, 26, 27, or 28. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 24.

[0035] In some embodiments, the immunomodulatory portion is fused to the C-terminus of the targeting portion. In some embodiments, the immunomodulatory portion is fused to the N-terminus of the targeting portion.

[0036] In some embodiments, the targeting portion is an antibody comprising a light chain and a heavy chain, and the immunomodulatory portion is fused to the C-terminus of the heavy chain of the targeting portion.

[0037] In some embodiments, the targeting portion is an antibody comprising a light chain and a heavy chain, and the immunomodulatory portion is fused to the C-terminus of the light chain of the targeting portion.

[0038] In some embodiments, the targeted portion is an antibody that specifically binds to hEGFR, comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the heavy chain or the light chain via a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0039] In some embodiments, the targeted portion is an antibody that specifically binds to hEGFR, comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the light chain via a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24.

[0040] In some embodiments, the targeted portion comprises an antibody comprising a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0041] In some embodiments, the liquid pharmaceutical composition is sterile.

[0042] In one embodiment, a liquid pharmaceutical composition is provided herein, comprising: (a) a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII; (b) about 5 mM to about 20 mM citrate-phosphate buffer; and (c) about 6% by weight / vol. to about 10% by weight / vol. sucrose, wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 6.5.

[0043] In some embodiments, the targeted portion comprises an antibody comprising a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0044] In some embodiments, the fusion protein is present at a concentration of approximately 25 mg / ml.

[0045] In some embodiments, the liquid pharmaceutical composition further comprises about 0.01 to 0.05% by weight / volume of polysorbate 20.

[0046] In one embodiment, a liquid pharmaceutical composition is provided herein, comprising: (a) a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII; (b) about 10 mM citrate phosphate buffer; and (c) about 8% by weight / volume of sucrose, wherein the liquid pharmaceutical composition has a pH of about 6.0.

[0047] In some embodiments, the pharmaceutical composition further comprises about 0.02% by weight / volume of polysorbate 20.

[0048] In some embodiments, the targeted portion comprises an antibody comprising a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0049] In some embodiments, the fusion protein is present at a concentration of approximately 25 mg / ml.

[0050] In one embodiment, a liquid pharmaceutical composition is provided herein, comprising: (a) a fusion protein of about 25 mg / mL comprising a targeting moiety and an immunomodulatory moiety, wherein the targeting moiety comprises an antibody comprising a heavy chain having an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain having an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29; (b) about 10 mM citrate phosphate buffer; (c) about 8% by weight / volume of sucrose; and (d) about 0.02% by weight / volume of polysorbate 20, wherein the liquid pharmaceutical composition has a pH of about 6.0.

[0051] In one embodiment, a method for treating human cancer in a subject having cancer is provided herein, the method comprising the step of administering to the subject a liquid pharmaceutical composition described herein.

[0052] In some embodiments, the liquid pharmaceutical composition is administered in an amount effective to treat the cancer.

[0053] In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 2000 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 20 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 30 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 40 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 50 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 100 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 900 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 800 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 700 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 800 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 700 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 600 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 500 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 400 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 300 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 100 mg. In some embodiments, the fusion protein is administered to the human subject in a dose of approximately 10 mg to 50 mg.

[0054] In some embodiments, the fusion protein is administered to the human subject in doses of approximately 50 mg, 60 mg, 64 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, the fusion protein is administered to the human subject in doses of approximately 64 mg, 240 mg, 800 mg, or 1600 mg.

[0055] In some embodiments, the fusion protein is administered to the human subject every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the fusion protein is administered to the human subject every week.

[0056] In some embodiments, the fusion protein is administered to the human subjects for three weeks.

[0057] In some embodiments, the administration step includes intravenous injection of the liquid pharmaceutical composition.

[0058] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is metastatic, recurrent, and / or refractory, or any combination thereof.

[0059] In some embodiments, the cancer comprises cancer cells that include genomic amplification of the EGFR gene, which can be detected, for example, by biopsy or fluorescence in situ hybridization.

[0060] In some embodiments, the cancer comprises cancer cells with genomic modifications in the KRAS gene. In some embodiments, the modification in the KRAS gene is a G12D substitution. In some embodiments, the modification in the KRAS gene is a G13D modification.

[0061] In some embodiments, the cancer is selected from the group consisting of eye, stomach, colon, rectum, colorectal, breast, anal, pancreatic, thyroid, liver, ovarian, lung, skin, brain, spinal cord, head, and neck cancers.

[0062] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is squamous cell lung cancer (SqCLC). In some embodiments, the SqCLC comprises cancer cells that do not express a detectable level of programmed death ligand 1, as measured by biopsy. In some embodiments, the SqCLC comprises cancer cells that include genomic amplification of the EGFR gene, as detected, for example, by biopsy or fluorescence in situ hybridization.

[0063] In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is RAS wild-type microsatellite-stable colorectal cancer (RAS WT MSS CRC). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is spinal cord cancer. In some embodiments, the spinal cord cancer is chordoma. In some embodiments, the cancer is eye cancer. In some embodiments, the eye cancer is ocular melanoma. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is thyroid cancer. In some embodiments, the thyroid cancer is undifferentiated thyroid carcinoma (ATC). In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is recurrent HNSCC. In some embodiments, the cancer is metastatic HNSCC. In some embodiments, the cancer is recurrent and metastatic HNSCC. In some embodiments, the cancer is squamous cell carcinoma of the anal canal (SCCAC). In some embodiments, the cancer is recurrent SCCAC. In some embodiments, the cancer is metastatic SCCAC. In some embodiments, the cancer is recurrent and metastatic SCCAC.

[0064] In one embodiment, a method for preparing a liquid pharmaceutical composition is provided herein, comprising the steps of (a) culturing mammalian cells in which one or more nucleic acids encoding a fusion protein comprising a targeting moiety and an immunomodulatory moiety are stably incorporated into their genome in a cell culture medium such that the cells secrete the fusion protein into the cell culture medium, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII; (b) purifying the fusion protein from the cell culture medium; and (c) preparing the pharmaceutical composition described herein. [Brief explanation of the drawing]

[0065] [Figure 1] This is a schematic diagram illustrating the development strategy for the formulations described herein. [Figure 2] This is a schematic diagram illustrating the pH screening study described in Example 1, as well as the BCA101 formulations tested at pH 5.0, 5.5, 6.0, and 6.5. [Figure 3] This is a scatter plot showing the percentage of high molecular weight proteins (HMWPs) in the bulk tangential flow filtration composition (TFF) and the final drug product (FDP) at each test pH (5.0, 5.5, 6.0, and 6.5). [Figure 4] This is a scatter plot showing the percentage of low monomeric proteins in bulk TFF composition and FDP at each test pH (5.0, 5.5, 6.0, and 6.5). [Figure 5] This is a dot graph showing the percentage of low molecular weight proteins (LMWPs) in bulk TFF compositions and FDP at each test pH (5.0, 5.5, 6.0, and 6.5). [Figure 6-1] This is a series of line graphs showing the results of the differential scanning calorimetry (DSC) analysis described in Example 1 for each BCA101 test formulation that was evaluated. [Figure 6-2] (Same as above.) [Figure 7] Figure 6 is a scatter plot summarizing the DSC analysis results described in Example 1 for each test formulation shown. [Figure 8] Figure 8A is a line graph showing the HMWP percentage at 40°C for test formulations with pH 6.0 and 6.5. Figure 8B is a dot graph showing the HMWP percentage slope per week at 40°C for test formulations with pH 6.0 and 6.5. [Figure 9] Figure 9A is a line graph showing the monomer percentage at 40°C for the test formulations with pH 6.0 and 6.5. Figure 9B is a dot graph showing the monomer percentage slope per week at 40°C for the test formulations with pH 6.0 and 6.5. [Figure 10] Figure 10A is a line graph showing the LMWP percentage at 40°C for test formulations with pH 6.0 and 6.5. Figure 10B is a dot graph showing the LMWP percentage slope per week at 40°C for test formulations with pH 6.0 and 6.5. [Figure 11] This is a schematic diagram showing the process of the buffer screening study described in Example 1 and the composition of the evaluated test formulation. [Figure 12] Figure 12A is a line graph showing the results of DSC analysis for a citrate buffer preparation with a pH of 6.0. Figure 12B is a line graph showing the results of DSC analysis for a citrate buffer preparation with a pH of 6.5. [Figure 13] Figure 13A is a line graph showing the results of DSC analysis for a succinate buffer preparation with a pH of 6.0. Figure 13B is a line graph showing the results of DSC analysis for a succinate buffer preparation with a pH of 6.5. [Figure 14] Figure 14A is a line graph showing the results of DSC analysis for a histidine buffer preparation with a pH of 6.0. Figure 14B is a line graph showing the results of DSC analysis for a histidine buffer preparation with a pH of 6.5. [Figure 15]Figure 15A is a line graph showing the results of DSC analysis for a citrate phosphate buffer preparation with a pH of 6.0. Figure 15B is a line graph showing the results of DSC analysis for a citrate phosphate buffer preparation with a pH of 6.5. [Figure 16] Figures 12A to 15B are dot graphs showing an overview of the DSC analysis data. [Figure 17] This is a schematic diagram illustrating the process of the tension modifier screening described in Example 1, which involves testing a test formulation containing sucrose or trehalose. [Figure 18] This is a photograph of the BCA101 fusion protein in a formulation containing 25 mg / ml of BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0), comparing the color of the BCA101 liquid formulation to the pharmacopoeia (Ph.Eu.2.2.2) color standard solution. [Figure 19] This table shows the absorbance at 506 nm of the BCA101 fusion protein in formulations containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate-phosphate buffer (pH 6.0) from toxicology study batches, internal reference standard (IRS) batches, and dose range discovery batches. [Figure 20] This is a photographic copy of the BCA101 fusion protein in a preparation containing 25 mg / ml of BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0), comparing the clarity and turbidity of the BCA101 liquid preparation to the pharmacopoeia standard (formazin suspension, Ph.Eu. 2.2.1). [Figure 21]This table shows the turbidity in turbidity units (NTU) of the BCA101 fusion protein in a formulation containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate-phosphate buffer (pH 6.0). Batches analyzed before filtration are indicated as "BF," and batches analyzed after filtration are indicated as "AF." The assay was performed according to Ph.Eur.2.2.1, the entire contents of which are incorporated herein by reference. [Figure 22] This is a series of dot graphs showing the pH, gravimetric osmolality, protein concentration, and bifunctional capacity of BCA101 active pharmaceutical ingredient (DS) formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over a 24-month period stored in a 5 mL Celsius bag at -20°C. Samples were analyzed at time 1, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 23] This is a series of dot graphs showing the pH, gravimetric osmolality, protein concentration, and bifunctional capacity of BCA101 drug products (DP) formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over a 24-month period stored in a 5 mL Celsius bag at -20°C. Samples were analyzed at time 1, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 24] This table shows a comparison of long-term stability data for BCA101 DS, presented in Figure 22, and DP, presented in Figure 23. [Figure 25]This is a series of dot graphs showing the LMWP percent, monomer percent, and HMWP percent of BCA101 DS formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over a 24-month period stored in a 5 mL Celsius bag at -20°C. Samples were analyzed at time 1, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 26] This is a series of dot graphs showing the LMWP percent, monomer percent, and HMWP percent of BCA101 DP formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over a 24-month period stored in a 5 mL Celsius bag at -20°C. Samples were analyzed at time 1, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 27] This table shows a comparison of long-term stability data for BCA101 DS, presented in Figure 25, and DP, presented in Figure 26. [Figure 28] This figure shows an exemplary manufacturing process of the present disclosure for producing a BCA101 formulation described herein, for example, a formulation comprising 25 mg / ml of BCA101, 8.0 wt / vol% sucrose, 0.02 wt / vol% polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0). [Figure 29] This line graph shows the pH trend of BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5℃ for 24 months. [Figure 30] This line graph shows the trend in gravimetric osmolality of BCA100 active pharmaceutical ingredient (DS) during 24 months of storage at -20±5℃. [Figure 31]This line graph shows the trend in protein concentration data for BCA100 active pharmaceutical ingredient (DS) during 24 months of storage at -20±5℃. [Figure 32] This line graph shows the trend in high molecular weight protein percentages of BCA100 active pharmaceutical ingredient (DS) stability data during 24 months of storage at -20±5℃. [Figure 33] This line graph shows the trend in monomer protein percentage of BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5℃ for 24 months. [Figure 34] This line graph shows the trend in the percentage of low molecular weight proteins in the BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5℃ for 24 months. [Figure 35] This line graph shows the trend of the percentage of total protein at the pre-peak of BCA100 active pharmaceutical ingredient (DS) stability data during 24 months of storage at -20±5℃. [Figure 36] This line graph shows the trend of the percentage of total protein in the post-peak of BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5℃ for 24 months. [Figure 37] This line graph shows the trend of the percentage of total protein at the main peak of the BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5℃ for 24 months. [Figure 38] This line graph shows the trend of relative potency in BCA100 active pharmaceutical ingredient (DS) stability data measured by a bifunctional ELISA during storage at -20±5°C for 24 months. [Figure 39] This line graph shows the trend of relative potency in BCA100 active pharmaceutical ingredient (DS) stability data during storage at -20±5°C for 24 months, as measured by an inhibitory growth (IOP) assay. [Figure 40] This line graph shows the pH trend of BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months. [Figure 41]This line graph shows the trend in gravimetric osmolality of BCA100 drug product (DP) stability data over 24 months at 5±3℃. [Figure 42] This line graph shows the trend in protein concentration data for BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months. [Figure 43] This line graph shows the trend in sample volume for BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months. [Figure 44] This line graph shows the trend in high molecular weight protein percentages in BCA100 drug product (DP) stability data during 24 months of storage at 5±3℃. [Figure 45] This line graph shows the trend in monomer protein percentage data for BCA100 drug product (DP) stability data during 24 months of storage at 5±3℃. [Figure 46] This line graph shows the trend in the percentage of low molecular weight proteins in BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months. [Figure 47] This line graph shows the trend of the percentage of total protein at the pre-peak of BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months. [Figure 48] This line graph shows the trend in the percentage of total protein in the post-peak of BCA100 drug product (DP) stability data during 24 months of storage at 5±3℃. [Figure 49] This line graph shows the trend in the percentage of total protein at the main peak of the BCA100 drug product (DP) stability data during 24 months of storage at 5±3℃. [Figure 50] This line graph shows the trend in relative potency of BCA100 drug product (DP) stability data during storage at 5±3℃ for 24 months, as measured by a bifunctional ELISA. [Figure 51]This line graph shows the trend in relative potency of BCA100 drug product (DP) stability data during storage at 5±3°C for 24 months, as measured by an inhibitory growth (IOP) assay. [Figure 52] These are iCE electrophoresis images of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (upper panel) and BCA101 DS GF19000040 (lower panel). The pI markers on the left and right correspond to 4.05 and 8.4, respectively. [Figure 53] The intact mass spectra of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (upper panel) and BCA101 DS GF19000040 (lower panel), obtained using MALDI-TOF, are shown. [Figure 54] The UV chromatograms of peptides produced from trypsin digestion of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (upper panel) and BCA101 DS batch GF19000040 (lower panel) are shown. [Figure 55] The MS2 tandem spectrum of the N-terminus of the heavy chain of BCA101 DS batch GF19000040 is shown. [Figure 56] The MS2 tandem spectrum of the C-terminus of the heavy chain of BCA101 DS batch GF19000040 is shown. [Figure 57] The MS2 tandem spectrum of the N-terminus of the light chain of BCA101 DS batch GF19000040 is shown. [Figure 58] The MS2 tandem spectrum of the C-terminus of the linker from BCA101 DS batch GF19000040 is shown. [Figure 59] The MS spectrum of the C-terminus of the TGFβRII chain from BCA101 DS batch GF19000040 is shown. [Figure 60] This shows the overlay of the far-UV CD spectrum of BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS. [Figure 61]This shows the overlay of the near-UV CD spectrum of BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS. [Figure 62] The UV chromatogram profile of the trypsin-unreduced peptide from BCA101 DS batch GF19000040 is shown along with BL.14.0901 / R / 17 / 021 / F DS. [Figure 63] This shows an overlay of the NP-HPLC profile of N-glycans from BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS. The N-glycans printed here were identified by MS. Identification of "other species" is in progress. [Figure 64] The RP-HPLC chromatograms of sialic acid estimation for BCA101 DS batch GF19000040 are shown, with overlays of NGNA, NANA standards, and corresponding buffer blanks. [Modes for carrying out the invention]

[0066] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that individual publications, patents, or patent applications are specifically and individually incorporated by reference.

[0067] overview This disclosure provides, in particular, pharmaceutical formulations of the bifunctional fusion proteins described herein that enable long-term storage of the preparations without unacceptable protein instability (e.g., degradation) or loss of bifunctional activity. In some embodiments, one functional aspect of the fusion protein (e.g., a targeting moiety) has an acidic pI, while a second functional aspect (e.g., an immunomodulatory domain) has a basic pI. In some embodiments, the hEGFR fusion protein comprises a targeting moiety that specifically binds to hEGFR and an immunomodulatory moiety that includes the amino acid sequence of the extracellular domain of hTGFβRII. The pharmaceutical compositions disclosed herein may be particularly useful in the treatment of cancers activated by hEGFR.

[0068] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the claimed subject matter pertains. It should be understood that the above general descriptions and the following detailed descriptions are illustrative and descriptive only and do not limit any claimed subject matter. In this application, the use of singular forms includes plural forms unless otherwise indicated.

[0069] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise clearly indicated by the context. Furthermore, the use of the term “including,” and other forms such as “include,” “includes,” and “included,” is not limited.

[0070] Where an aspect is described herein using the word “comprising,” it is understood that similar aspects are also provided, which are otherwise described using the terms “consisting of” and / or “essentially consisting of.”

[0071] The section headings used herein are for structural purposes only and should not be considered to limit the subject matter described herein.

[0072] When used herein, the term "and / or" is to be interpreted as a specific disclosure that each of the two specified characteristics or components may or may not be associated with the other. Accordingly, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which this disclosure relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary for many of the terms used herein.

[0074] Units, prefixes, and symbols are shown in the form recognized by the International System of Units (SI). Numerical ranges include the number defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure that can be obtained by reference to this specification as a whole. Thus, the terms defined immediately following are more fully defined by reference to this specification as a whole.

[0075] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integers within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0076] The terms “approximately” or “essentially composed of” mean a value or composition that is within the acceptable margin of error for a particular value or composition as determined by those skilled in the art, which will in part depend on how that value or composition is measured or determined, i.e., the limitations of the measuring system. For example, “approximately” or “essentially composed of” may mean within 1 or a standard deviation greater than 1, according to convention in the art. Alternatively, “approximately” or “essentially composed of” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, this term may mean a value of up to one order of magnitude or up to five times the value. Where a particular value or composition is provided in this application and claims, unless otherwise indicated, the meaning of “approximately” or “essentially composed of” shall be assumed to be within the acceptable margin of error for that particular value or composition.

[0077] The terms “subject” and “patient” are used interchangeably herein and include any human or non-human animal. The term “non-human animal” includes, but is not limited to, vertebrates, e.g., non-human primates, sheep, dogs, and rodents, e.g., mice, rats, and guinea pigs. In some embodiments, the subject is human.

[0078] As used herein, the term “administer” means the physical delivery of a therapeutic agent (or a precursor of a therapeutic agent, which is metabolized or modified within the subject’s body to produce a therapeutic agent in vivo) to a subject using any of the various methods and delivery systems known to those skilled in the art. Illustrative routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, e.g., by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Therapeutic agents may be administered via parenteral routes or orally. Other parenteral routes of administration include topical, cutaneous, or mucosal administration routes, such as intranasal, intravaginal, intrarectal, sublingual, or topical. Administration may also be carried out, for example, as a single dose, multiple doses, and / or over a long period of one or more doses.

[0079] The terms “cancer” and “tumor” are used interchangeably herein and refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth, division and growth, lead to the formation of malignant tumors that can invade adjacent tissues and further metastasize to distal parts of the body through the lymphatic system or bloodstream.

[0080] The “therapeutic effective dose” of a therapeutic agent is any amount of the therapeutic agent, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or promotes disease regression, which is indicated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional impairment or disability resulting from disease distress. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the drug in in vitro assays.

[0081] The term “antibody” is used herein in its broadest sense and includes fully assembled antibodies, functional antibody fragments and functional variants capable of binding to antigens (e.g., Fab, F(ab')2, Fv, single-strand variable fragments (scFv), single-domain antibodies (e.g., VHH), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.), as well as non-antibody fragments that bind to antigens (e.g., recombinant fibronectin domains), and recombinant polypeptides including those mentioned above. Unless otherwise indicated, references to the numbering of specific amino acid residue positions in antibodies follow the EU numbering system described in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) ("Kabat"), and this entire content is incorporated herein by reference.

[0082] As used herein, the term “variable region” refers to a domain of the antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the natural antibody heavy and light chains (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions and three complementarity-determining regions.

[0083] As used herein, the term “complementarity-determining region” refers to each region of the antibody variable domain whose sequence is hypervariable and forms a structurally defined loop (“hypervariable loop”). Generally, a naturally quadruple antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). CDRs are described by Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) (“Kabat”) and Chothia et al., J Mol Biol 196:901-917 (1987), and this definition includes overlaps or subsets of amino acid residues when compared to one another. In any case, the application of any definition referring to an antibody's CDR is intended to fall within the scope of the definitions and terms used herein. Those skilled in the art can conventionally determine which residues constitute a particular CDR based on the variable region amino acid sequence of an antibody. Unless otherwise specified, CDR is defined according to the Kabat system.

[0084] As used herein, the term “fusion protein” and its grammatical equivalents refer to a protein comprising amino acid sequences derived from at least two distinct proteins. The amino acid sequences of the at least two distinct proteins may be directly linked through peptide bonds or operably linked through amino acid linkers. Thus, the term fusion protein encompasses embodiments such as, for example, a protein A-protein B, in which the amino acid sequence of protein A is directly linked to the amino acid sequence of protein B through peptide bonds, and embodiments such as a protein A-linker-protein B, in which the amino acid sequence of protein A is operably linked to the amino acid sequence of protein B through an amino acid linker.

[0085] As used herein, the term “fuse” and its grammatical equivalent refer to the operable connection of an amino acid sequence from one protein to an amino acid sequence from another protein. The term “fuse” encompasses both direct connection of two amino acid sequences through peptide bonds and indirect connection through amino acid linkers.

[0086] As used herein, the term “modified” in relation to nucleic acid sequences refers to a nucleic acid sequence that includes at least one substitution, addition, or deletion of nucleotides compared to a reference nucleic acid sequence. As used herein, the term “modified” in relation to amino acid sequences refers to an amino acid sequence that includes at least one substitution, addition, or deletion of amino acid residues compared to a reference amino acid sequence. Naturally occurring amino acid derivatives are not considered modified amino acids for the purpose of determining the percentage of identity between two amino acid sequences. For example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue is not considered an amino acid modification for the purpose of determining the percentage of identity between two amino acid sequences. Furthermore, for example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue would not be considered an amino acid “modified” as defined herein. Modifications may include the inclusion of non-natural amino acid residues.

[0087] The terms “identical” or “percent identity” in relation to nucleic acid sequences or amino acid sequences refer to the fact that at least two nucleic acid sequences or at least two amino acid sequences or subsequences have a specified percentage of identical nucleotides or amino acids when compared and aligned for the greatest match, as measured using a sequence comparison algorithm or by visual inspection. In sequence comparison, typically one sequence functions as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified if necessary, and program parameters for the sequence algorithm are specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Examples of suitable algorithms for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The percentage of identity between two sequences can be determined, with or without gap tolerance, using techniques similar to those described above. When calculating the percentage of identity, typically only perfect matches are counted. As mentioned above, the percentage of identity is based on amino acid matching in the smaller of the two proteins.

[0088] A “stable” pharmaceutical composition is one in which the proteins within it inherently maintain physical and / or chemical stability and / or biological activity during processing (e.g., ultrafiltration, diafiltration, other filtration steps, vial filling), transport, and / or storage of the active pharmaceutical ingredient and / or drug product containing the fusion protein described herein. Furthermore, the physical, chemical, and biological stability of the proteins in the formulation embodies the “stability” of the protein formulation, e.g., the fusion protein formulation, which is specific to the conditions under which the formulated drug product (DP) is stored.

[0089] A protein retains its "physical stability" in a pharmaceutical composition if, when measured by visual inspection of color and / or clarity, or by UV light scattering or size exclusion high-performance liquid chromatography, or by other suitable methods, it shows minimal signs of changes in secondary and / or tertiary structure (i.e., essential structure), or aggregation, and / or sedimentation and / or denaturation. Physical instability of a protein, i.e., loss of physical stability, can be caused by the formation of dimers and higher-order aggregates, invisible and visible particles, and oligomer formation resulting in sedimentation. The degree of physical degradation can be determined using various techniques depending on the type of degradation product of interest. Dimers and higher-order soluble aggregates can be quantified using size exclusion chromatography, while invisible particles can be quantified using light scattering, light shielding, or other suitable techniques.

[0090] A protein retains its "chemical stability" in a pharmaceutical composition if, at a given point in time, its chemical stability does not result in the formation or destruction of covalent bonds and does not lead to a change in the primary structure of the protein components, such as the fusion protein described herein. A change in primary structure can result in alterations of the secondary and / or tertiary and / or quaternary structure of the protein, and can result in the formation of aggregates or the inversion of already formed aggregates. Typical chemical alterations include isomerization, deamination, N-terminal cyclization, skeletal hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, beta-elimination, disulfide formation, disulfide scrambling, disulfide cleavage, and other changes resulting in alterations of primary structure, including D-amino acid formation. Chemical instability, i.e., loss of chemical stability, can be investigated by a variety of techniques, including ion-exchange chromatography, capillary isoelectric focusing, peptide digestion analysis, and several types of mass spectrometry. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Chemical modifications may include size alterations (e.g., clipping) which can be evaluated using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization mass spectrometry / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical modifications include charge alterations (e.g., resulting from deamidation) which can be evaluated by charge-based methods, such as, but not limited to, ion-exchange chromatography, capillary isoelectric focusing, or peptide mapping.

[0091] Loss of physical and / or chemical stability can result in changes in biological activity, either as an increase or decrease in the desired biological activity, depending on the modified and modified protein. A protein retains its “biological activity” in a pharmaceutical composition if its biological activity at a given point in time is within 30% of the biological activity shown at the time the pharmaceutical formulation was prepared. Activity is considered decreased if it falls below 70% of its initial value. Biological assays can include both in vivo and in vitro assays, e.g., ligand binding, potency, cell proliferation, or other surrogate measures of its biopharmaceutical activity. As an example, the biological activity of BCA101 described herein can be estimated using an ELISA that measures its binding ability to both hEGFR and hTGFβ. Briefly, to perform a bifunctional ELISA, a plate coated with recombinant hEGFR Fc was blocked and subsequently incubated with BCA101 for approximately 1 hour, and then incubated with recombinant hTGFβ1. hTGFβ1 bound to the hTGFβRII ECD portion of BCA101 was then detected using a biotinylated anti-hTGFβ1 antibody, followed by streptavidin-HRP. This ensures that a signal is obtained only if both arms are intact.

[0092] Pharmaceutical composition In certain embodiments, a pharmaceutical composition (e.g., a liquid pharmaceutical composition) comprising a fusion protein (e.g., a fusion protein described herein), a buffer, and a tonicity modifier, and having a pre-selected pH, is described herein. In some embodiments, the pharmaceutical composition comprises one or more further agents, including, for example, a surfactant. In some embodiments, the pharmaceutical composition is in liquid or powder form. In some embodiments, the pharmaceutical composition is in liquid form. In some embodiments, the pharmaceutical composition is particularly suitable for intravenous administration to a subject (e.g., a human subject).

[0093] buffer solution In some embodiments, the buffer is citrate phosphate buffer, citrate buffer, succinate buffer, or histidine buffer. In some embodiments, the buffer is citrate phosphate buffer. In some embodiments, the buffer is citrate buffer. In some embodiments, the buffer is succinate buffer. In some embodiments, the buffer is histidine buffer.

[0094] In some embodiments, the buffer concentration is approximately 5 mM to 40 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 25 mM, 5 mM to 20 mM, 5 mM to 15 mM, 5 mM to 10 mM, or 10 mM to 30 mM. In some embodiments, the buffer concentration is approximately 5 mM to 15 mM. In some embodiments, the buffer concentration is approximately 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM. In some embodiments, the buffer concentration is approximately 10 mM.

[0095] In certain embodiments, the buffer is a citrate phosphate buffer present at concentrations of approximately 5 mM to approximately 30 mM, 5 mM to approximately 25 mM, 5 mM to approximately 20 mM, 5 mM to approximately 15 mM, 5 mM to approximately 10 mM, or 10 mM to approximately 30 mM. In some embodiments, the buffer is a citrate phosphate buffer present at concentrations of approximately 5 mM to approximately 15 mM. In some embodiments, the buffer is a citrate phosphate buffer present at concentrations of approximately 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM. In some embodiments, the buffer is a citrate phosphate buffer present at a concentration of approximately 10 mM. In some embodiments, the pharmaceutical composition has a pH of approximately 6.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of approximately 6.0.

[0096] pH In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 8.0. In some embodiments, the pharmaceutical composition has a pH of about 5.5 to about 7.0, 6.0 to about 7.0, 5.5 to about 6.5, 5.5 to about 6.0, or 6.0 to about 6.5. In some embodiments, the pharmaceutical composition has a pH of about 6.0 to about 6.5. In some embodiments, the pharmaceutical composition has a pH of about 5.5, 6.0, 6.5, or 7.0. In some embodiments, the pharmaceutical composition has a pH of about 6.0. In some embodiments, the pharmaceutical composition has a pH of about 6.5.

[0097] Tension adjuster In some embodiments, the pharmaceutical composition contains a tonicity modifier such that the formulation has a final osmolality by weight of about 200 to 400 mOsmol / kg. In some embodiments, the pharmaceutical composition contains a tonicity modifier such that the formulation has a final osmolality by weight of about 250 to 350 mOsmol / kg. In some embodiments, the pharmaceutical composition contains a tonicity modifier such that the formulation has a final osmolality by weight of about 300 mOsmol / kg.

[0098] In some embodiments, the tension modifier comprises sucrose, trehalose, sorbitol, mannitol, or glycerol. In some embodiments, the tension modifier is sucrose. In some embodiments, the tension modifier is trehalose. In some embodiments, the tension modifier is present in concentrations of about 5 wt / vol% to about 10 wt / vol%, 6 wt / vol% to about 10 wt / vol%, 7 wt / vol% to about 10 wt / vol%, 8 wt / vol% to about 10 wt / vol%, 5 wt / vol% to about 9 wt / vol%, 5 wt / vol% to about 8 wt / vol%, 6 wt / vol% to about 9 wt / vol%, 6 wt / vol% to about 8 wt / vol%, 7 wt / vol% to about 9 wt / vol%, or 7 wt / vol% to about 8 wt / vol%. In some embodiments, the tension modifier is present in concentrations of about 5 wt / vol% to about 8 wt / vol%. In some embodiments, the tension modifier is present at concentrations of about 5% by weight / vol, 6% by weight / vol, 7% by weight / vol, 8% by weight / vol, 9% by weight / vol, or 10% by weight / vol. In some embodiments, the tension modifier is present at a concentration of about 8% by weight / vol.

[0099] In some embodiments, the tonic modifier is sucrose at a concentration such that the formulation has a final weight osmolality of about 300 mOsmol / kg. In some embodiments, the tonic modifier includes sucrose at concentrations of about 5 wt / vol% to about 10 wt / vol%, 6 wt / vol% to about 10 wt / vol%, 7 wt / vol% to about 10 wt / vol%, 8 wt / vol% to about 10 wt / vol%, 5 wt / vol% to about 9 wt / vol%, 5 wt / vol% to about 8 wt / vol%, 6 wt / vol% to about 9 wt / vol%, 6 wt / vol% to about 8 wt / vol%, 7 wt / vol% to about 9 wt / vol%, or 7 wt / vol% to about 8 wt / vol%. In some embodiments, the tonic modifier includes sucrose at concentrations of about 5 wt / vol% to about 8 wt / vol%. In some embodiments, the tension modifier is present at concentrations of about 5% by weight / vol, 6% by weight / vol, 7% by weight / vol, 8% by weight / vol, 9% by weight / vol, or 10% by weight / vol. In some embodiments, the tension modifier is sucrose at a concentration of about 8% by weight / vol.

[0100] surfactant In some embodiments, the pharmaceutical composition includes a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is polysorbate, polyethylene glycol dodecyl ether, poloxamer, 4-(l,l,3,3-tetramethylbutyl)phenyl-polyethylene glycol, alkyl saccharides and alkyl glycosides, Brij® 35 (i.e., polyethylene glycol dodecyl ether), poloxamer (i.e., polyethylene-polypropylene glycol, polyoxyethylene-polyoxypropylene block copolymer, poly(ethylene oxide-co-polypropylene oxide)), e.g., poloxamer 188 (i.e., Pluronic F68), or Triton® X-100 (i.e., 4-(l,l,3,3-tetramethylbutyl)phenyl-polyethylene glycol)). Exemplary polysorbates include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the surfactant is polysorbate 20.

[0101] In some embodiments, the surfactant concentration is approximately 0.005-0.1 wt / vol%, 0.01-0.1 wt / vol%, 0.02-0.1 wt / vol%, 0.01-0.9 wt / vol%, 0.01-0.8 wt / vol%, 0.01-0.7 wt / vol%, 0.01-0.6 wt / vol%, 0.01-0.5 wt / vol%, 0.01-0.4 wt / vol%, 0.01-0.3 wt / vol%, 0.01-0.2 wt / vol%, 0.01-0.1 wt / vol%, 0.02-0.9 wt / vol%, 0.02-0.8 wt / vol%, 0.02-0.7 wt The values ​​are volt / volume%, 0.02-0.6 weight / volume%, 0.02-0.5 weight / volume%, 0.02-0.4 weight / volume%, 0.02-0.3 weight / volume%, 0.02-0.2 weight / volume%, 0.02-0.1 weight / volume%, 0.005-0.9 weight / volume%, 0.005-0.8 weight / volume%, 0.005-0.7 weight / volume%, 0.005-0.6 weight / volume%, 0.005-0.5 weight / volume%, 0.005-0.4 weight / volume%, 0.005-0.3 weight / volume%, 0.005-0.2 weight / volume%, or 0.005-0.1 weight / volume%. In some embodiments, the concentration of the surfactant is about 0.01 wt / vol%, 0.02 wt / vol%, 0.03 wt / vol%, 0.04 wt / vol%, 0.05 wt / vol%, 0.06 wt / vol%, 0.07 wt / vol%, 0.08 wt / vol%, 0.09 wt / vol%, or 0.1 wt / vol%. In some embodiments, the concentration of the surfactant is about 0.02 wt / vol%.

[0102] In some embodiments, the surfactant is present in concentrations of approximately 0.005-0.1 wt / vol%, 0.01-0.1 wt / vol%, 0.02-0.1 wt / vol%, 0.01-0.9 wt / vol%, 0.01-0.8 wt / vol%, 0.01-0.7 wt / vol%, 0.01-0.6 wt / vol%, 0.01-0.5 wt / vol%, 0.01-0.4 wt / vol%, 0.01-0.3 wt / vol%, 0.01-0.2 wt / vol%, 0.01-0.1 wt / vol%, 0.02-0.9 wt / vol%, 0.02-0.8 wt / vol%, 0.02-0.7 wt / vol%, and 0. Polysorbate 20 with concentrations of 0.2-0.6 wt / vol.%, 0.02-0.5 wt / vol.%, 0.02-0.4 wt / vol.%, 0.02-0.3 wt / vol.%, 0.02-0.2 wt / vol.%, 0.02-0.1 wt / vol.%, 0.005-0.9 wt / vol.%, 0.005-0.8 wt / vol.%, 0.005-0.7 wt / vol.%, 0.005-0.6 wt / vol.%, 0.005-0.5 wt / vol.%, 0.005-0.4 wt / vol.%, 0.005-0.3 wt / vol.%, 0.005-0.2 wt / vol.%, or 0.005-0.1 wt / vol. In some embodiments, the surfactant is polysorbate 20 at a concentration of about 0.01 wt / vol%, 0.02 wt / vol%, 0.03 wt / vol%, 0.04 wt / vol%, 0.05 wt / vol%, 0.06 wt / vol%, 0.07 wt / vol%, 0.08 wt / vol%, 0.09 wt / vol%, or 0.1 wt / vol%. In some embodiments, the surfactant is polysorbate 20 at a concentration of about 0.02 wt / vol%.

[0103] Osmolality In some embodiments, the pharmaceutical composition has a weight osmolality of about 100 mOsmol / kg to about 400 mOsmol / kg, about 150 mOsmol / kg to about 400 mOsmol / kg, 150 mOsmol / kg to about 350 mOsmol / kg, 150 mOsmol / kg to about 300 mOsmol / kg, 200 mOsmol / kg to about 400 mOsmol / kg, 250 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 400 mOsmol / kg, 300 mOsmol / kg to about 350 mOsmol / kg, 250 mOsmol / kg to about 350 mOsmol / kg, or 250 mOsmol / kg to about 300 mOsmol / kg. In some embodiments, the pharmaceutical composition has a weight osmolality of about 250 mOsmol / kg to about 350 mOsmol / kg. In some embodiments, the pharmaceutical composition has a weight osmolality of about 250 mOsmol / kg, 300 mOsmol / kg, or 350 mOsmol / kg. In some embodiments, the pharmaceutical composition has a weight osmolality of about 300 mOsmol / kg.

[0104] Exemplary functional characteristics Stability and storage The pharmaceutical composition may be stored in any suitable container known to those skilled in the art, such as a bag (e.g., Celsius® bag, Flexboy® bag) or a glass vial (e.g., USP 10R glass vial). Containers for appropriate storage at various temperatures (e.g., -80°C, -20°C, 2–8°C) are known to those skilled in the art and may be appropriately selected. In some embodiments, when stability is measured at -20°C, the pharmaceutical composition is stored in a Celsius® bag or a Flexboy® bag. In some embodiments, when stability is measured at 2–8°C, the pharmaceutical composition is stored in a glass vial.

[0105] In some embodiments, the pharmaceutical composition exhibits increased stability for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months compared to a reference pharmaceutical composition when refrigerated or frozen. In some embodiments, the pharmaceutical composition exhibits increased chemical stability for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months compared to a reference pharmaceutical composition when refrigerated or frozen. In some embodiments, the pharmaceutical composition exhibits increased physical stability for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months compared to a reference pharmaceutical composition when refrigerated or frozen.

[0106] In some embodiments, the pharmaceutical composition is stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -80°C. In some embodiments, the pharmaceutical composition is stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the pharmaceutical composition is stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2-8°C.

[0107] In some embodiments, the pharmaceutical composition is chemically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is chemically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -80°C. In some embodiments, the pharmaceutical composition is chemically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is chemically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the pharmaceutical composition is chemically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2-8°C.

[0108] In some embodiments, the pharmaceutical composition is physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -80°C. In some embodiments, the pharmaceutical composition is physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the pharmaceutical composition is physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2-8°C.

[0109] In some embodiments, the pharmaceutical composition is chemically and physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the pharmaceutical composition is chemically and physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -80°C. In some embodiments, the pharmaceutical composition is chemically and physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the pharmaceutical composition is chemically and physically stable for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2-8°C.

[0110] In some embodiments, the pharmaceutical composition is stable through at least one, two, three, four, or five freeze-thaw cycles, each comprising a 48-hour freeze cycle at -80°C or -20°C, and a thaw cycle comprising 4 hours of thawing at 25°C in an incubator. In some embodiments, the pharmaceutical composition is chemically stable through at least one, two, three, four, or five freeze-thaw cycles, each comprising a 48-hour freeze cycle at -80°C or -20°C, and a thaw cycle comprising 4 hours of thawing at 25°C in an incubator. In some embodiments, the pharmaceutical composition is physically stable through at least one, two, three, four, or five freeze-thaw cycles, each comprising a 48-hour freeze cycle at -80°C or -20°C, and a thaw cycle comprising 4 hours of thawing at 25°C in an incubator. In some embodiments, the pharmaceutical composition is chemically and physically stable through at least one, two, three, four, or five freeze-thaw cycles, the freeze-thaw cycle comprising a 48-hour freeze cycle at -80°C or -20°C, and the thaw cycle comprising a 4-hour thaw cycle at 25°C in an incubator.

[0111] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -80°C. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2-8°C.

[0112] In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -80°C for 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -20°C for 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months. In some embodiments, the concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at 2-8°C for 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months.

[0113] In some embodiments, the pharmaceutical composition has a shelf life of at least 12 months, 24 months, 36 months, or 48 months when stored at -80°C. In some embodiments, the pharmaceutical composition has a shelf life of at least 12 months, 24 months, 36 months, or 48 months when stored at -20°C. In some embodiments, the pharmaceutical composition has a shelf life of at least 12 months, 24 months, 36 months, or 48 months when stored at 2-8°C.

[0114] In some embodiments, the pharmaceutical composition contains the fusion protein in aggregate form in amounts less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the pharmaceutical composition contains the fusion protein in aggregate form in amounts less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the pharmaceutical composition contains the fusion protein in aggregate form in amounts less than 5%. In some embodiments, the pharmaceutical composition contains the fusion protein in aggregate form in approximately 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains the fusion protein in aggregate form in approximately 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% after storage at 2-8°C for at least 12 months, 24 months, or 36 months.

[0115] In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, or not exceeding 20%. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, or not exceeding 10%. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, or not exceeding 5%. In some embodiments, the pharmaceutical composition contains fusion proteins in aggregate form at a concentration of no more than 5%. In some embodiments, the pharmaceutical composition contains fusion proteins in aggregate form at a concentration of no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, or no more than 20% after storage at -80°C for at least 12, 24, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, or not exceeding 10% after storage at -80°C for at least 12, 24, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, or not exceeding 5% after storage at -80°C for at least 12, 24, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 5% after storage at -80°C for at least 12, 24, or 36 months.In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, or not exceeding 20%. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, not exceeding 20%, or not exceeding 30% after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, or not exceeding 10%. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, not exceeding 20%, or not exceeding 30% after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, not exceeding 20%, or not exceeding 30%. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, or not exceeding 5% after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 5%.In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, not exceeding 20%, or not exceeding 30% after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, not exceeding 10%, or not exceeding 20% ​​after storage at 2-8°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, not exceeding 5%, not exceeding 6%, not exceeding 7%, not exceeding 8%, not exceeding 9%, or not exceeding 10% after storage at 2-8°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 1%, not exceeding 2%, not exceeding 3%, not exceeding 4%, or not exceeding 5% after storage at 2-8°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains aggregated fusion proteins in amounts not exceeding 5% after storage at 2-8°C for at least 12 months, 24 months, or 36 months. Aggregation can be measured by any suitable method known in the art, including that described in Example 1 of this disclosure. Aggregation can be evaluated, for example, by size exclusion chromatography (SEC).

[0116] Stability can be measured by any assay known to those skilled in the art, including the one described in Example 1 of this disclosure. Various analytical techniques for measuring protein stability are discussed, for example, in Wang, W. (1999), Instability, stabilization and formulation of liquid protein pharmaceuticals, Int J Pharm 185: 129-188. Stability can be measured at a selected temperature for a selected period of time (e.g., 1 week, 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months).

[0117] Dual functionality of fusion proteins The fusion proteins described herein have two distinct functions: 1) specifically binding to hEGFR, and 2) specifically binding to hTGFβ. In some embodiments, the fusion proteins retain their bifunctional activity for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when refrigerated or frozen. In some embodiments, the fusion proteins retain their bifunctional activity for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at -20°C. In some embodiments, the fusion proteins retain their bifunctional activity for at least 3 months, 6 months, 12 months, 18 months, 24 months, or 36 months when stored at 2–8°C.

[0118] In some embodiments, the fusion proteins described herein retain at least 95%, 96%, 97%, 98%, 99%, or 100% of their hEGFR binding activity (e.g., as measured by ELISA). In some embodiments, the fusion proteins described herein retain 95%, 96%, 97%, 98%, 99%, or 100% of their hTGFβ binding activity (e.g., as measured by ELISA). In some embodiments, the fusion proteins described herein retain at least 95%, 96%, 97%, 98%, 99%, or 100% of their hEGFR binding activity (e.g., as measured by ELISA), and retain at least 96%, 97%, 98%, 99%, or 100% of their hTGFβ binding activity (e.g., as measured by ELISA).

[0119] In some embodiments, the fusion proteins described herein lose less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of their hEGFR binding activity (e.g., as measured by ELISA). In some embodiments, the fusion proteins described herein lose less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of their hTGFβ binding activity (e.g., as measured by ELISA). In some embodiments, the fusion proteins described herein lose less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of their hEGFR binding activity (e.g., as measured by ELISA), and lose less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of their hTGFβ binding activity (e.g., as measured by ELISA).

[0120] The dual functionality of the fusion proteins described herein can be evaluated by methods known in the art, including those described in Example 1 of this disclosure. For example, the dual functionality of the fusion proteins described herein can be evaluated by two separate or combined ELISA assays for both functionalities of the fusion proteins described herein (i.e., 1) specific binding to hEGFR and 2) specific binding to hTGFβ.

[0121] Fusion protein In a particular embodiment, a pharmaceutical composition comprising a multifunctional fusion protein having a targeting moiety and an immunomodulatory moiety is provided herein, wherein (i) the targeting moiety comprises a polypeptide that specifically binds to a membrane-bound target protein and has a basic isoelectric point (pI), and (ii) the immunomodulatory moiety comprises a polypeptide that specifically binds to a soluble target protein having an acidic pI, and the membrane-bound target protein and the soluble target protein are different.

[0122] In a particular embodiment, a pharmaceutical composition comprising a multifunctional (bifunctional) fusion protein having a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII, is provided herein.

[0123] hEGFR targeting moiety In some embodiments, the hEGFR-targeting moiety comprises an antibody, or a functional fragment or functional variant thereof. In some embodiments, the antibody is a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, F(v), a single-domain antibody, a single-chain antibody, or VHH.

[0124] In some embodiments, the anti-hEGFR antibody is selected from the group consisting of cetuximab and panitumumab. In some embodiments, the anti-hEGFR antibody is a functional fragment of cetuximab and panitumumab. In some embodiments, the anti-hEGFR antibody is a functional variant of cetuximab and panitumumab.

[0125] Cetuximab In some embodiments, the anti-hEGFR antibody is cetuximab. In some embodiments, the anti-hEGFR antibody cross-competes with cetuximab. In some embodiments, the anti-hEGFR antibody binds to the same epitope as cetuximab. In some embodiments, the anti-hEGFR antibody has the same CDR as cetuximab.

[0126] In some embodiments, the anti-hEGFR antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-hEGFR antibody comprises VH containing VH CDR1, which contains the amino acid sequence of SEQ ID NO: 1 having 0, 1, 2, or 3 amino acid modifications; VH CDR2, which contains the amino acid sequence of SEQ ID NO: 2 having 0, 1, 2, or 3 amino acid modifications; and / or VH CDR3, which contains the amino acid sequence of SEQ ID NO: 3 having 0, 1, 2, or 3 amino acid modifications.

[0127] In some embodiments, the anti-hEGFR antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2 having one, two, or up to three amino acid modifications; and / or VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 3 having one, two, or up to three amino acid modifications.

[0128] In some embodiments, the anti-hEGFR antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-hEGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 having 0, 1, 2, or 3 amino acid modifications, VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 having 0, 1, 2, or 3 amino acid modifications, and / or VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 having 0, 1, 2, or 3 amino acid modifications.

[0129] In some embodiments, the anti-hEGFR antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-hEGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence of SEQ ID NO: 4 with one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 5 with one, two, or three amino acid modifications; and / or VL CDR3 containing the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 6 with one, two, or three amino acid modifications.

[0130] In some embodiments, the anti-hEGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 1 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2 having 0, 1, 2, or 3 amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 having 0, 1, 2, or 3 amino acid modifications.

[0131] In some embodiments, the anti-hEGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 having one, two, or three amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 4 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 5 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 having one, two, or three amino acid modifications.

[0132] In some embodiments, the anti-hEGFR antibody comprises VH including VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3.

[0133] In some embodiments, the anti-hEGFR antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6.

[0134] In some embodiments, the anti-hEGFR antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. The anti-hEGFR antibody comprises VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. The VL includes a VL containing a VL CDR3 that has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 6.

[0135] In some embodiments, the anti-hEGFR antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-hEGFR antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-hEGFR antibody comprises VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, and VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0136] In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-hEGFR antibody comprises a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0137] In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0138] Panitumumab In some embodiments, the anti-hEGFR antibody is panitumumab. In some embodiments, the anti-hEGFR antibody cross-competes with panitumumab. In some embodiments, the anti-hEGFR antibody binds to the same epitope as panitumumab. In some embodiments, the anti-hEGFR antibody has the same CDR as panitumumab.

[0139] In some embodiments, the anti-hEGFR antibody comprises a variable heavy chain (VH) containing three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3. In some embodiments, the anti-hEGFR antibody comprises VH containing VH CDR1, which contains the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 amino acid modifications; VH CDR2, which contains the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2, or 3 amino acid modifications; and / or VH CDR3, which contains the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2, or 3 amino acid modifications.

[0140] In some embodiments, the anti-hEGFR antibody comprises VH CDR1 containing the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13 having one, two, or three amino acid modifications; and / or VH CDR3 containing the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 14 having one, two, or three amino acid modifications.

[0141] In some embodiments, the anti-hEGFR antibody comprises a variable light chain (VL) containing three complementarity-determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-hEGFR antibody comprises a VL containing VL CDR1 containing the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 amino acid modifications, VL CDR2 containing the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2, or 3 amino acid modifications, and / or VL CDR3 containing the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 amino acid modifications.

[0142] In some embodiments, the anti-hEGFR antibody comprises a VL containing VL CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15 having one, two, or three amino acid modifications; VL CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 16 having one, two, or three amino acid modifications; and / or VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 having one, two, or three amino acid modifications.

[0143] In some embodiments, the anti-hEGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2, or 3 amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2, or 3 amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 amino acid modifications.

[0144] In some embodiments, the anti-hEGFR antibody comprises VH, which includes VH CDR1 containing the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 12 having one, two, or three amino acid modifications; VH CDR2 containing the amino acid sequence of SEQ ID NO: 13 or the amino acid sequence of SEQ ID NO: 13 having one, two, or three amino acid modifications; and VL, which includes VL CDR1 containing the amino acid sequence of SEQ ID NO: 15 or the amino acid sequence of SEQ ID NO: 15 having one, two, or three amino acid modifications; VL CDR2 containing the amino acid sequence of SEQ ID NO: 16 or the amino acid sequence of SEQ ID NO: 16 having one, two, or three amino acid modifications; and VL CDR3 containing the amino acid sequence of SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 17 having one, two, or three amino acid modifications.

[0145] In some embodiments, the anti-hEGFR antibody comprises VH, which includes VH CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12; VH CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13; and VH CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0146] In some embodiments, the anti-hEGFR antibody comprises VLs including VL CDR1 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, VL CDR2 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and VL CDR3 containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.

[0147] In some embodiments, the anti-hEGFR antibody comprises VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13, and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14; and the anti-hEGFR antibody comprises VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. The VL includes a VL containing CDR3, which has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2 and SEQ ID NO: 17.

[0148] In some embodiments, the anti-hEGFR antibody contains VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-hEGFR antibody contains VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-hEGFR antibody comprises VH which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, and VL which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0149] In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-hEGFR antibody comprises a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0150] In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0151] In some embodiments, the anti-hEGFR antibody includes the antibodies listed in Table 1.

[0152] In some embodiments, the anti-hEGFR antibody comprises VH including VH CDR1 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 1, VH CDR2 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 1, and VH CDR3 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 1.

[0153] In some embodiments, the anti-hEGFR antibody comprises VLs including VL CDR1 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 1, VL CDR2 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR2 in Table 1, and VL CDR3 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 1.

[0154] In some embodiments, the anti-hEGFR antibody includes VH CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR1 in Table 1, VH CDR2 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR2 in Table 1, and VH CDR3 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH CDR3 in Table 1, VL CDR1 containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR1 in Table 1, and VL CDR1 in Table 1. The VL includes VLs containing VL CDR2 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2, and VL CDR3 having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL CDR3 in Table 1.

[0155] In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the heavy chain in Table 1. In some embodiments, the anti-hEGFR antibody comprises a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the light chain in Table 1. In some embodiments, the anti-hEGFR antibody comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the heavy chain in Table 1, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the light chain in Table 1.

[0156] In some embodiments, the anti-hEGFR antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 1. In some embodiments, the anti-hEGFR antibody comprises VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the antibody in Table 1. In some embodiments, the anti-hEGFR antibody comprises VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VH of the antibody in Table 1, and VL having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VL of the antibody in Table 1.

[0157] [Table 1-1]

[0158] [Table 1-2]

[0159] [Table 1-3]

[0160] hTGFβ trap In a particular embodiment, the fusion protein comprises a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain (ECD) of hTGFβRII.

[0161] In some embodiments, hTGFβRII ECD binds to at least one hTGFβ isoform. In some embodiments, hTGFβRII ECD binds to hTGFβ1. In some embodiments, hTGFβRII ECD binds to hTGFβ3. In some embodiments, hTGFβRII ECD does not bind to hTGFβ2.

[0162] In some embodiments, the hTGFβRII ECD contains a sequence of naturally occurring hTGFβRII ECD sufficient to allow the protein to bind to hTGFβ. In some embodiments, the hTGFβRII ECD contains a sequence of naturally occurring TGFβRII ECD sufficient to allow the protein to bind to hTGFβ1. In some embodiments, the hTGFβRII ECD contains a sequence of naturally occurring hTGFβRII ECD sufficient to allow the protein to bind to hTGFβ3.

[0163] In some embodiments, the extracellular domain of hTGFβRII includes a shortened portion of SEQ ID NO: 23, which enables binding to hTGFβ. The extracellular domain of hTGFβRII may be shortened at the N-terminus, C-terminus, or both the N-terminus and C-terminus. The shortening may involve the deletion of 1 to 10 amino acids. The shortening may involve the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The shortening may involve the deletion of 1, 2, 3, 4, or 5 amino acids from the N-terminus, C-terminus, or both the N-terminus and C-terminus.

[0164] In some embodiments, the extracellular domain of hTGFβRII contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the extracellular domain of hTGFβRII consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the extracellular domain of hTGFβRII consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23.

[0165] [Table 2]

[0166] Orientation In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the targeting portion. In some embodiments, the immunomodulatory portion is operably connected to the N-terminus of the targeting portion.

[0167] In some embodiments, the targeting portion is an antibody (or its functional fragment or variant) comprising 1) VH or a heavy chain and 2) VL or a light chain. In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the VH or heavy chain. In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the VL or light chain. In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the constant region of the heavy chain. In some embodiments, the immunomodulatory portion is operably connected to the C-terminus of the constant region of the light chain. In some embodiments, the immunomodulatory portion is operably connected to the N-terminus of the VH or heavy chain. In some embodiments, the immunomodulatory portion is operably connected to the N-terminus of the VL or light chain.

[0168] Linker In some embodiments, the targeting and immunomodulatory portions of the fusion protein are directly operably connected. In some embodiments, the targeting and immunomodulatory portions of the fusion protein are indirectly operably connected. In some embodiments, the targeting and immunomodulatory portions of the fusion protein are indirectly operably connected via a linker. In some embodiments, the linker is a peptide linker.

[0169] Any suitable peptide linker known in the art can be used, enabling the immunomodulatory and targeting portions to bind to their respective antigens. Exemplary peptide linkers, including glycine and serine amino acids, are provided in Table 3.

[0170] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences of SEQ ID NOs. 24-28. In some embodiments, the linker includes any one of the amino acid sequences of SEQ ID NOs. 24-28, or any one of the amino acid sequences of SEQ ID NOs. 24-28 having one, two, or three amino acid modifications.

[0171] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 having one, two, or three amino acid modifications.

[0172] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25 having one, two, or three amino acid modifications.

[0173] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 26, or the amino acid sequence of SEQ ID NO: 26 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 26, or the amino acid sequence of SEQ ID NO: 26 having one, two, or three amino acid modifications.

[0174] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 27, or the amino acid sequence of SEQ ID NO: 27 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 27, or the amino acid sequence of SEQ ID NO: 27 having one, two, or three amino acid modifications.

[0175] In some embodiments, the linker includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker includes an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence of SEQ ID NO: 28 having one, two, or three amino acid modifications. In some embodiments, the linker essentially consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker consists of the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence of SEQ ID NO: 28 having one, two, or three amino acid modifications.

[0176] [Table 3]

[0177] Exemplary fusion protein Exemplary fusion proteins of this disclosure are provided in Table 4.

[0178] In one embodiment, the fusion protein contains BCA101.

[0179] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0180] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0181] In some embodiments, the fusion protein comprises a heavy chain, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises a light chain, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 10, and a light chain, the amino acid sequence of which comprises the amino acid sequence of SEQ ID NO: 29.

[0182] In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a light chain containing the amino acid sequence of SEQ ID NO: 29 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10 having one, two, or three amino acid modifications, and a light chain containing the amino acid sequence of SEQ ID NO: 29 having one, two, or three amino acid modifications.

[0183] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0184] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0185] In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a light chain consisting of the amino acid sequence of SEQ ID NO: 29 having one, two, or three amino acid modifications. In some embodiments, the fusion protein comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10 having one, two, or three amino acid modifications, and a light chain consisting of the amino acid sequence of SEQ ID NO: 29 having one, two, or three amino acid modifications.

[0186] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0187] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0188] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0189] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0190] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0191] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0192] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0193] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0194] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0195] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0196] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0197] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 21, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0198] In some embodiments, the fusion protein includes a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the fusion protein includes a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein comprises a heavy chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34, and a light chain containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0199] In some embodiments, the fusion protein includes a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the fusion protein includes a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34, and a light chain consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0200] [Table 4-1]

[0201] [Table 4-2]

[0202] [Table 4-3]

[0203] [Table 4-4]

[0204] [Table 4-5]

[0205] In some embodiments, the pharmaceutical composition contains the fusion protein described herein in concentrations of approximately 5-50 mg / ml, 5-40 mg / ml, 5-30 mg / ml, 5-25 mg / ml, 10-50 mg / ml, 20-50 mg / ml, 25-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml, 25-50 mg / ml, 25-40 mg / ml, or 25-30 mg / ml. In some embodiments, the pharmaceutical composition contains the fusion protein described herein in concentrations of approximately 20-30 mg / ml. In some embodiments, the pharmaceutical composition contains the fusion protein described herein in concentrations of approximately 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In some embodiments, the pharmaceutical composition contains the fusion protein described herein at a concentration of about 25 mg / ml.

[0206] In some embodiments, the fusion protein is BCA101 at concentrations of approximately 50 mg / ml, 5–40 mg / ml, 5–30 mg / ml, 5–25 mg / ml, 10–50 mg / ml, 20–50 mg / ml, 25–50 mg / ml, 20–50 mg / ml, 20–40 mg / ml, 20–30 mg / ml, 25–50 mg / ml, 25–40 mg / ml, or 25–30 mg / ml. In some embodiments, the fusion protein is BCA101 and is present at a concentration of approximately 20–30 mg / ml. In some embodiments, the fusion protein is BCA101 and is present at concentrations of approximately 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In some embodiments, the fusion protein is BCA101, present at a concentration of approximately 25 mg / ml.

[0207] In some embodiments, the fusion protein comprises a targeting moiety and an immunomodulatory moiety, wherein the targeting moiety comprises an antibody comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29, and is present in the pharmaceutical composition at a concentration of about 20-30 mg / ml. In some embodiments, the fusion protein comprises a targeting moiety and an immunomodulatory moiety, wherein the targeting moiety comprises an antibody comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29, and is present in the pharmaceutical composition at a concentration of about 25 mg / ml.

[0208] Manufacturing method In some embodiments, methods for producing the pharmaceutical compositions described herein are provided herein. In some embodiments, the method comprises the steps of: culturing mammalian cells containing one or more nucleic acids encoding the fusion protein described herein in a cell culture medium such that the cells secrete the fusion protein into the cell culture medium; purifying the fusion protein from the cell culture medium; and preparing the pharmaceutical composition described herein.

[0209] In some embodiments, cells have one or more nucleic acids encoding the fusion protein stably incorporated into their genome. In some embodiments, cells have one or more nucleic acids encoding the fusion protein transiently incorporated into the cell. In some embodiments, one or more nucleic acids encoding the fusion protein are introduced into the cell by transfection or transduction. Methods of transfection and transduction are well known in the art.

[0210] Any suitable mammalian cell can be used for the expression of the fusion protein. For example, well-known mammalian cells include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), human fetal kidney line (293 cells or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. 36: 59)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216, e.g., DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), and African green monkey kidney cells (VERO-76, ATCC Examples include, but are not limited to, CRL-1587, human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et al, 1982, Annals NY Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatocellular carcinoma lineage (Hep G2).

[0211] The host cells used to produce the fusion proteins described herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimum Essential Medium ((MEM), (Sigma-Aldrich Co.), RPML 1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma-Aldrich Co.), are suitable for culturing host cells. In addition, see Ham et al, 1979, Meth. Enz. 58: 44, Barnes et al, 1980, Anal. Biochem. 102: 255, any of the media described in one or more of U.S. Patents 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, International Publication No. 90 / 103430, and International Publication No. 87 / 00195 may be used as a culture medium for host cells, the entire contents of which are incorporated herein by reference.

[0212] Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), small amounts of elements (defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Other supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, are those previously used in host cells selected for expression and will be apparent to those skilled in the art.

[0213] The fusion protein secreted from the cells can be purified using any suitable method known in the art. Examples include size exclusion chromatography, hydroxyl apatite chromatography, affinity chromatography, gel electrophoresis, dialysis, or tangential flow filtration. In some embodiments, the fusion protein or pharmaceutical composition is subjected to sterile filtration. In some embodiments, the pharmaceutical composition is produced as an active pharmaceutical ingredient and subjected to sterile filtration to produce a drug product.

[0214] therapeutic use In one embodiment, a method for treating cancer in a subject is provided herein, which involves administering a pharmaceutical composition described herein to a subject having cancer.

[0215] In some embodiments, the methods disclosed herein are used as an alternative to standard treatment. In certain embodiments, standard treatment is used in combination with any of the methods disclosed herein. Standard treatments for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Network (NCCN), a coalition of 21 major cancer centers in the United States, publishes the NCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®), which provide detailed and up-to-date information on standard treatment procedures for a wide range of cancers. In some embodiments, the methods disclosed herein are used after standard treatment has failed.

[0216] Exemplary cancer In some embodiments, cancer is metastatic. In some embodiments, cancer is recurrent. In some embodiments, cancer is both metastatic and recurrent.

[0217] In some embodiments, the cancer is activated by EGFR. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy.

[0218] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is metastatic, recurrent, and / or refractory, or any combination thereof.

[0219] In some embodiments, the cancer comprises cancer cells that contain genomic amplification of the EGFR gene, which is detected, for example, by biopsy or fluorescence in situ hybridization.

[0220] In some embodiments, the cancer comprises cancer cells that contain genomic modifications in the KRAS gene. In some embodiments, the modification in the KRAS gene is a G12D substitution. In some embodiments, the modification in the KRAS gene is a G13D modification.

[0221] In some embodiments, the cancer is selected from the group consisting of eye, stomach, colon, rectum, colorectal, breast, anal, pancreatic, thyroid, liver, ovarian, lung, skin, brain, spinal cord, head, and neck cancers.

[0222] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is squamous cell lung cancer (SqCLC). In some embodiments, the SqCLC comprises cancer cells that do not express detectable levels of programmed death ligand 1, measured by biopsy. In some embodiments, the SqCLC comprises cancer cells that contain genomic amplification of the EGFR gene, which is detected, for example, by biopsy or fluorescence in situ hybridization.

[0223] In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is RAS wild-type microsatellite-stable colorectal cancer (RAS WT MSS CRC). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC).

[0224] In some embodiments, the cancer is spinal cord cancer. In some embodiments, the spinal cord cancer is chordoma. In some embodiments, the cancer is eye cancer. In some embodiments, the eye cancer is ocular melanoma. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma.

[0225] In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is thyroid cancer. In some embodiments, the thyroid cancer is undifferentiated thyroid cancer (ATC). In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is stomach cancer.

[0226] In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is recurrent HNSCC. In some embodiments, the cancer is metastatic HNSCC. In some embodiments, the cancer is metastatic and recurrent HNSCC. In some embodiments, the cancer is anal canal. In some embodiments, the cancer is squamous cell carcinoma of the anal canal (SCCAC). In some embodiments, the cancer is recurrent SCCAC. In some embodiments, the cancer is metastatic SCCAC. In some embodiments, the cancer is metastatic and recurrent SCCAC.

[0227] Example medication regimens and schedules In some embodiments, the fusion protein (i.e., a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII) is administered to a subject with cancer at a therapeutically effective dose. In some embodiments, the fusion protein is administered to a subject with cancer at a fixed dose. In some embodiments, the fusion protein is administered to a subject with cancer at a constant dose. In some embodiments, the fusion protein is administered to a subject with cancer at a weight-based dose.

[0228] In several appropriate amounts, the fusion protein is approximately 50mg-2000mg, 100mg-2000mg, 150mg-2000mg, 200mg-2000mg, 300mg-2000mg, 400mg-2000mg, 500mg-2000mg, 600mg-2000mg, 700mg-2000mg, 800mg-2000mg, 9000mg-20 The fusion protein is administered to the subject in doses of 00 mg, 1000 mg to 2000 mg, 1500 mg to 2000 mg, 50 mg to 100 mg, 50 mg to 500 mg, 50 mg to 400 mg, 50 mg to 300 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 500 mg, 100 mg to 400 mg, 100 mg to 300 mg, or 100 mg to 200 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 200 mg to 2000 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 50 mg, 60 mg, 64 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 64 mg, 240 mg, 800 mg, or 1600 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 64 mg. In some embodiments, the fusion protein is administered to the subject in doses of approximately 240 mg. In some embodiments, the fusion protein is administered to the subject in a dose of approximately 800 mg. In some embodiments, the fusion protein is administered to the subject in a dose of approximately 1600 mg.

[0229] In some embodiments, the fusion protein is administered to the subject every week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks. In some embodiments, the fusion protein is administered to the subject every week. In some embodiments, the fusion protein is administered to the subject every two weeks. In some embodiments, the fusion protein is administered to the subject every three weeks. In some embodiments, the fusion protein is administered to the subject every four weeks. In some embodiments, the fusion protein is administered to the subject every five weeks. In some embodiments, the fusion protein is administered to the subject every six weeks.

[0230] kit In one embodiment, a kit comprising a liquid pharmaceutical composition described herein for therapeutic use is provided herein. The kit typically includes a label indicating the intended use of the contents of the kit, and instructions for use. The term "label" includes any descriptive or documentary material provided on or with the kit, or accompanying the kit. Accordingly, this disclosure provides a kit for treating a subject with cancer, comprising (a) a certain dosage of a pharmaceutical composition described herein, and (b) instructions for use in a method of treatment disclosed herein. In a particular embodiment for treating a human patient, the kit comprises a liquid pharmaceutical composition described herein, comprising BCA101.

[0231] The present invention is further illustrated by the following embodiments, which are not to be construed as further limitations. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Examples]

[0232] [Example 1] Development of BCA101 formulations The objective of this study was to develop a formulation of a bifunctional fusion protein in which the two functional domains of the protein have different isoelectric points. This study utilizes BCA101 as such a fusion protein. BCA101 is a bifunctional fusion protein comprising an anti-hEGFR antibody and the extracellular domain of hTGFβRII fused to the C-terminus of the anti-hEGFR antibody light chain, as described herein. The anti-hEGFR antibody domain of BCA101 has a basic pI (isoelectric point), while the hTGFβRII extracellular domain has an acidic pI. Therefore, it was necessary to develop a formulation capable of accommodating a fusion protein containing two proteins with different functions, structures, and pIs. The formulation would need to maintain the physicochemical stability, functional and biological efficacy of the fusion protein during long-term storage (e.g., 12-24 months) at refrigerated (e.g., 2-8°C) or frozen (e.g., -20°C) temperatures. The formulation was developed through a series of studies shown in Figure 1.

[0233] pH screening research To determine the pH at which BCA101 is most stable, a pH screening study was performed. Ultrafiltration of BCA101 (approximately 35 mg / ml) was performed using tangential flow filtration (TFF) at pH 5.0, 5.5, 6.0, and 6.5. The filtration product was formulated with 10 mM citrate phosphate buffer (10 mM), 0.02 wt / vol% polysorbate 20, and 25 mg / ml BCA101 (Figure 2). The percentages of high molecular weight proteins (HMWP) (Figure 3), protein monomers (Figure 4), and low molecular weight proteins (LMWP) (Figure 5) were measured by size exclusion chromatography for the bulk tangential flow filtration composition (TFF) and the final drug product (FDP). The stability of BCA101 in each pH formulation was further analyzed by differential scanning calorimetry (DSC). The DSC line graphs for each formulation are shown in Figure 6, and an overview of the data at each pH is shown in Figure 7 and Table 5.

[0234]

Table 5

[0235] The percentages of HMWP at 40 °C (Figures 8A - 8B), monomers (Figures 9A - 9B), and LMWP (Figures 10A - 10B) were further determined for the formulations at pH 6.0 and pH 6.5. Based on the above data, the formulations at pH 6.0 and 6.5 were selected for further development.

[0236] Buffer screening research Four different buffers (citric acid, succinic acid, histidine, and citrate phosphate (citric acid monohydrate (0.573 mg / mL), disodium hydrogen phosphate dihydrate (1.294 mg / mL))) were tested at the selected pHs of 6.0 and 6.5. The drug product was stored at 2 - 8 °C in 2R USP Type 1 glass vials with 13 mm gray-coated FluroTec® rubber stoppers and flip-off seals. Each formulation contained 25 mg / ml of BCA101, 0.02 wt / vol% of polysorbate 20, and 10 mM of the test buffer (citric acid, succinic acid, histidine, and citrate phosphate) (Figure 11). The stability of BCA101 was measured by DSC for each formulation: citric acid buffer (Figures 12A - 12B), succinic acid buffer (Figures 13A - 13B), histidine buffer (Figures 14A - 14B), and citrate phosphate buffer (Figures 15A - 15B). An overview of the DSC data across the four test buffers is presented in Figure 16.

[0237] Each formulation was further evaluated for physical appearance, filterability, Tm (by DSC), and HMWP (stress stability) (Table 6). Based on the above data, citrate phosphate (pH 6.0) and succinic acid (pH 6.5) were selected for further development.

[0238]

Table 6

[0239] Tension Modifier Screening The tonic modifiers were screened for each of the selected buffers and pH levels described above. Each test formulation contained 25 mg / ml of BCA101, 0.02 wt / wt%, or 5.0 wt / vol% of the tonic modifier (sucrose or trehalose), and 10 mM buffer (citric acid phosphate (pH 6.0) or succinic acid (pH 6.5)) according to Table 7 below (Figure 17).

[0240] [Table 7]

[0241] Stress stability research A stress stability test was performed at 40°C, followed by a freeze-thaw study involving three freezing cycles in a cryovial, each consisting of 48 hours of freezing at -80°C and -20°C, and 4 hours of thawing in an incubator at 25°C. The results of the freeze-thaw and stress stability studies are summarized in Table 8. No changes were observed in pH, gravimetric osmolality, or protein concentration in any of the test formulations during either the freeze / thaw study or the stress study.

[0242] [Table 8]

[0243] The gravimetric osmolality of sucrose formulations was evaluated (Table 9). A concentration of 5 wt / volume yielded gravimetric osmolality values ​​in the range of 170–240 mOsmol / kg. The sucrose concentration was further optimized based on achieving a target gravimetric osmolality of 300 mOsmol / kg. In the absence of sucrose, the gravimetric osmolality of the BCA101 formulation with 0.02 wt / volume polysorbate-20 and 10 mM citrate phosphate buffer ranged from 30–35 mOsmol / kg. A sucrose concentration of 8.0 wt / volume is considered to achieve 300 mOsmol / kg for the BCA101 drug product.

[0244] [Table 9]

[0245] Color and Transparency Studies The color of BCA101 preparations containing 25 mg / ml BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0) was evaluated by comparing it to the color standard solutions of the pharmacopoeia (Ph.Eu.2.2.2) (Figure 18). A table of reference standard solutions is provided in Tables 10-12 below. The absorbance at 506 nm of BCA101 batches (toxicology research batches, IRS, and DRF) is shown in Figure 19.

[0246] [Table 10]

[0247] [Table 11]

[0248] [Table 12]

[0249] The clarity and turbidity of BCA101 preparations containing 25 mg / ml BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0) were evaluated in comparison to the pharmacopoeia standard (formazin suspension, Ph.Eu. 2.2.1) (Figure 20). The reference standard solution is provided in Table 13 below. The NTU values ​​of the BCA101 samples are shown in Figure 21. The assay was performed according to Ph.Eur. 2.2.1, and its entirety is incorporated herein by reference.

[0250] [Table 13]

[0251] Long-term stability testing The stability of BCA101 in formulations containing 25 mg / ml BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0) was evaluated for toxicological study batches, with the active pharmaceutical ingredient (DS) in 5 mL Celsius bags at 2–20°C (Figures 22 and 25) and the drug product (DP) in glass vials at 2–8°C (Figures 23 and 26). The pH, gravimetric osmolality, protein concentration, and functionality of both arms of the BCA101 fusion protein (measured by a bifunctional ELISA measuring the ability to bind to hEGFR and hTGFβ) of the active pharmaceutical ingredient (API) (Figure 22) and the drug product (Figure 23) were evaluated over 24 months. Data points were acquired at baseline, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months. A graphical comparison of the results obtained from the long-term stability study between the API and the drug product is shown in Figure 24. Briefly, to perform the bifunctional ELISA, plates coated with recombinant hEGFR Fc were blocked and subsequently incubated with BCA101 for approximately 1 hour, and then incubated with recombinant hTGFβ1. hTGFβ1 bound to the hTGFβRII ECD portion of BCA101 was then detected with biotinylated anti-hTGFβ1 antibody, followed by streptavidin-HRP. This means that a signal will only be obtained when both arms are intact.

[0252] HMWP percent, monomer percent, and LMWP percent were also evaluated for toxicology study batches, with the active pharmaceutical ingredient (DS) in 5 mL Celsius bags at 2–20°C (Figure 25) and the drug product (DP) in glass vials at 2–8°C (Figure 26). A graphical comparison of the results obtained from long-term stability studies between the active pharmaceutical ingredient and the drug product is shown in Figure 27.

[0253] A diagrammatic representation of the exemplary formulation process described in the above-mentioned examples is shown in Figure 28.

[0254] [Example 2] Long-term storage stability of BCA101 active pharmaceutical ingredient (DS) The purpose of this study was to evaluate the long-term storage stability of BCA101 active pharmaceutical ingredient (DS) at -20±5°C for 24 months. Two different batches of BCA100 DS (BL.14.0901 / R / 17 / 021 F DS (R&D toxicology batch) and BS17006883 (GMP development batch)) were evaluated, each containing 25 mg / ml of BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0), stored at -20±5°C in either a Flexboy bag (BL.14.0901 / R / 17 / 021 F DS) or a Celsius FFT or Celsius Pak bag (BS17006883). pH, gravimetric osmolality, protein concentration, monomer percentage, high molecular weight protein (HMWP)%, low molecular weight protein (LMWP)%, functionality of both arms of the BCA101 fusion protein (measured by bifunctional ELISA for the ability to bind to hEGFR and hTGFβ), visual description, color, clarity, protein concentration, purity by SEC-HPLC and RP-HPLC, inhibition of EGFR-expressing cell proliferation, bacterial endotoxin, and number of contaminating microorganisms were evaluated over 24 months. Data points were generally taken at the start, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months (except those shown in Tables 14 and 15 below). Tables 14 and 15 provide a summary of stability data for both batches of BCA101 DS. Descriptive and trend data for each individual stability test are provided below and in Figures 29-39.

[0255] Table 14 below provides a summary of the long-term stability data for BCA101 DS batch BL.14.0901 / R / 17 / 021 F DS stored at -20±5℃, and individual stability tests are described in more detail below.

[0256] [Table 14]

[0257] Table 15 below provides a summary of the long-term stability data for BS17006883 (GMP batch) of BCA101 DS stored at -20±5℃, and individual stability tests are described in more detail below.

[0258] [Table 15-1]

[0259] [Table 15-2]

[0260] Visual observation Visual inspection is a qualitative evaluation of the description, clarity, and color of the manufactured active pharmaceutical ingredient. Based on the manufacturing data, the following specifications were set for BCA101 DS: Description: Absence of intervening foreign particles was set to "clear, cloudy liquid, free of foreign substances"; Clarity: "Turbidity not exceeding Pharmacopoeia Reference Standard III (NTU value is NMT 18 NTU)"; Color: "Not more strongly colored than Pharmacopoeia Reference Standard BY4". Batch BS17006883 of BCA101 DS was tested for visual inspection and showed to meet each of the specifications for up to 24 months from the date of manufacture (see Table 15). Batch BL.14.0901 / R / 17 / 021 F DS of BCA101 DS was not tested for visual inspection.

[0261] pH range The pH range for BCA101 DS was set to pH 6.00 ± 0.30 units, based on the optimal stability of BCA101 DS. Any pH change is an indicator of the degradation of one or more components in the formulation. Both BCA101 DS batches were tested and determined to meet the pH specifications for up to 24 months from the manufacturing date. Trend analysis of the DS batches showed no significant pH changes, as shown in Tables 14 and 15 and Figure 29.

[0262] Osmolality MMOLL concentration is one of the parameters, and for parenteral formulations, it needs to be controlled to approximate that of plasma to avoid adverse reactions associated with the injection of hypotonic / hypertonic DS during administration. The isosmolality of BCA101 is achieved primarily using sucrose in the formulation, and the specification for MMOLL concentration for BCA101 DS was set to 270–330 mOsmol / kg. Any change in sucrose concentration during the stability period can result in a change in MMOLL concentration, which indicates an effect on the stability of the substance. As shown in Figure 30 and Tables 14 and 15, the MMOLL concentration of the BL.14.0901 / R / 17 / 021 F DS BCA101 DS batch was within specifications for up to 24 months, and the BS17006883 BCA101 DS batch was also measured at 6 and 9 months and determined to be within specifications. The gravimetric osmolality of the BS17006883 BCA101 DS batch has not been tested beyond 9 months.

[0263] protein concentration Protein concentration provides information about the amount of DS in the sample. Based on development data, the limit for BCA101 DS protein concentration was set at 25.00 ± 2.00 mg / mL. As shown in Figure 31 and Tables 14 and 15, all BCA101 DS batches met the protein concentration specifications for up to 24 months from the manufacturing date.

[0264] Microbial count and bacterial endotoxin test (BET) The acceptable limits for BET and microbial contamination count for BCA101 DS were set at ≤0.25 EU / mg (NMT) and ≤10 CFU / 100 mL (NMT), respectively. As shown in Table 15, batch BS17006883 BCA101 DS met both the BET and microbial contamination count specifications for up to 24 months from the manufacturing date. Batch BL.14.0901 / R / 17 / 021 F DS BCA101 DS was not tested for either microbial contamination count or BET.

[0265] Purity measured by SEC-HPLC SEC-HPLC provides information on monomer content and associated HMWP. Acceptance criteria for BCA101 DS were set as follows: monomer % NLT 93.00%, HMWP % NMT 3.00%, and LMWP % reported results. As shown in Figure 32 (HMWP%), Figure 33 (monomer %), and Figure 34 (LMWP%), and in Tables 14 and 15, the SEC-HPLC purity results met the specification limits for both BCA101 DS batches for up to 24 months from the manufacturing date.

[0266] Purification by RP-HPLC RP-HPLC provides purity information regarding hydrophobic variants. Acceptance criteria for BCA101 DS were set as follows: all main peaks NLT 70.0%, all post-peaks NMT 24%, and all pre-peaks NMT 6%. As shown in Figure 35 (all pre-peaks), Figure 36 (all post-peaks), and Figure 37 (main peaks %), and in Tables 14 and 15, the RP-HPLC purity results for batch BS17006883 BCA101 DS met the specification limits for up to 24 months from the manufacturing date. Batch BL.14.0901 / R / 17 / 021 F DS BCA101 DS was not tested by RP-HPLC.

[0267] Bifunctional ELISA The co-binding efficacy of BCA101 to the EGF receptor (EGFR) and TGFβ1 ligand was determined using a bifunctional ELISA. Briefly, plates coated with recombinant hEGFR Fc were blocked and then incubated with BCA101 for approximately 1 hour, followed by incubation with recombinant hTGFβ1. hTGFβ1 bound to the hTGFβRII ECD portion of BCA101 was then detected with a biotinylated anti-hTGFβ1 antibody, followed by streptavidin-HRP. This ensures that a signal is obtained only if both antigen-binding arms of BCA101 (binding to EGFR and binding to the TGFβ1 ligand) are intact. Assay acceptance criteria were set at an average relative potency of 0.80–1.25 compared to a reference standard.

[0268] As shown in Figure 38, the relative potency of both BCA101 DS batches remained well within assay acceptance limits up to 24 months from the manufacturing date, which was the last time point tested.

[0269] Growth inhibition (IOP) assay The ability of BCA101 DS to inhibit FaDu cancer cell growth by binding to its target EGFR was determined using an Inhibition on Growth (IOP) assay. This assay provides a method for determining the number of viable cells in a culture by quantifying the amount of ATP present. Readings were based on the luminescence of luciferin catalyzed by luciferase in the presence of Mg2+, and the ATP released by viable cells. The assay tolerance was set at an average relative potency of 0.80–1.25 compared to a reference standard.

[0270] As shown in Figure 39 (and Table 14), the BS17006883 BCA101 DS batch was determined to meet the IOP standard of 0.80–1.25 for up to 24 months from the date of manufacture. The BL.14.0901 / R / 17 / 021 F DS BCA101 DS batch was not tested by IOP.

[0271] conclusion The data described above demonstrates that multiple batches of BCA101 API stored at -20±5°C for up to 24 months from the manufacturing date met the specifications for a variety of comprehensive critical quality attributes (release parameters). Based on stability trend analysis, no significant changes were observed in pH, gravimetric osmolality, SEC-HPLC, protein content / concentration, RP-HPLC, or functionality. Data obtained from R&D (BL.14.0901 / R / 17 / 021 F DS) and development GMP batches (BS17006883) demonstrate the physicochemical and functional stability of the BCA101 API formulation over 24 months from the manufacturing date when stored at -20±5°C in either Flexboy or Celsius FFT / Celsius Pak bags.

[0272] [Example 3] Long-term storage stability of BCA101 drug products (DP) The objective of this study was to evaluate the long-term storage stability of the BCA101 drug product (DP) stored at 5±3°C for 24 months. Two different batches of BCA100 DP (BL.14.0901 / R / 17 / 021 F DS (R&D batch) and BS18002245 (GMP development batch)) were evaluated, stored in 10R USP Type I clear glass vials at 5±3°C, containing 25 mg / ml BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0). pH, gravimetric osmolality, protein concentration, monomer percentage, high molecular weight protein (HMWP)%, low molecular weight protein (LMWP)%, functionality of both antigen-binding arms of the BCA101 fusion protein (measured by bifunctional ELISA for their ability to bind to hEGFR and hTGFβ), purity (by SEC-HPLC and RP-HPLC), inhibition of EGFR-expressing cell proliferation, visual indication, clarity, color, sample volume, seal integrity, particulate matter invisible to the naked eye, bacterial endotoxin, and sterility were evaluated over a 24-month period. Data points were generally taken at the start, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months (except as shown in Tables 16 and 17). Tables 16 and 17 provide a summary of stability data for the first and second batches of BCA101 DP. Individual stability test descriptions and trend data are provided below and in Figures 40–51.

[0273] Table 16 below provides a summary of the long-term stability data for BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch (R&D batch) stored at 5±3℃, and individual stability tests are described in more detail below.

[0274] [Table 16]

[0275] Table 17 below provides a summary of the long-term stability data for BS18002245 BCA101 DS batch (GMP batch) stored at 5±3℃, and individual stability tests are described in more detail below.

[0276] [Table 17-1]

[0277] [Table 17-2]

[0278] Visual observation Visual inspection is a qualitative assessment of the description, clarity, and color of the manufactured drug product. Based on manufacturing data, the specifications were set as follows: Description: Absence of intervening foreign particles was set to "clear, cloudy liquid, free of foreign substances"; Clarity: "Cloudiness not exceeding Pharmacopoeia Reference Standard III (NTU value NMT 18 NTU)"; Color: "Not more strongly colored than Pharmacopoeia Reference Standard BY4". Batch BS18002245 BCA101 DP was tested for visual inspection and showed to meet each of the specifications for up to 24 months from the date of manufacture (see Table 17). Batch BL.14.0901 / R / 17 / 021 F DP BCA101 DP was not tested for visual inspection.

[0279] pH range Based on the optimal stability of BCA101 DP, the pH range of BCA101 DP was set to pH 6.00 ± 0.30 units. Any pH change indicates the degradation of one or more components in the formulation. As shown in Figure 40 and Tables 16 and 17, all BCA101 DP batches met the pH specifications for up to 24 months from the filling / manufacturing date. Trend analysis of DS batches showed no significant changes in detected pH (Figure 40, Table 16, and Table 17).

[0280] Osmolality MMOLL concentration is one of the parameters, and for parenteral formulations, it needs to be controlled to approximate that of plasma to avoid adverse reactions associated with the injection of hypotonic / hypertonic DP during administration. The isosmolality of BCA101 DP was achieved primarily using sucrose, and the specification for MMOLL concentration was set to 270–330 mOsmol / kg. Any changes in sucrose concentration during the stability period may result in changes in MMOLL concentration, which indicate an effect on the stability of the protein. As shown in Figure 41 and Table 16, the MMOLL concentration of the BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was within specifications for up to 24 months from the submission / manufacturing date. The BS18002245 BCA101 DP batch was not tested for MMOLL concentration.

[0281] protein concentration Protein concentration provides information about the amount of BCA101 DP in the sample. Based on development data, the protein concentration limit was set at 25.00 ± 2.00 mg / mL. As shown in Figure 42 (and Tables 16 and 17), all batches of BCA101 DS met specifications for up to 24 months from the filling / manufacturing date.

[0282] Particles visible to the naked eye The standard for particles visible to the naked eye was set as follows: USP <790> Particles visible to the naked eye during injection and USP <l>Based on the injection (component solution), "the injectable preparation shall be substantially free of visible particles." BCA101 DP vials were manufactured in a controlled environment and tested for visible particles before batch release. No visible particles were observed at the time of batch release or throughout the stability study (24 months) (no changes were expected as the vials were aseptically sealed). Batch BS18002245 BCA101 DP met the specifications for up to 24 months from the manufacturing date (see Table 17). Batch BL.14.0901 / R / 17 / 021 F DP BCA101 DP was not tested for visible particles.

[0283] Particulate matter invisible to the naked eye Pharmacopoeia limits (USP) <788> There are two standards defined based on the size of particles invisible to the naked eye, based on the following: Particles invisible to the naked eye (10 μm or larger): 6000 or fewer particles per container (NMT); Particles invisible to the naked eye (25 μm or larger): 600 or fewer particles per container (NMT). The BS18002245 BCA101 DP batch met the standard for up to 24 months from the date of manufacture (see Table 17). The BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was not tested for particulate matter invisible to the naked eye.

[0284] Sterility testing and BET (bacterial endotoxin testing) The BET and sterility acceptance criteria are based on standard pharmacopoeia limits, which are specified as less than 0.25 EU / mg and "absence of microbial growth," respectively. Batch BS18002245 BCA101 DP stored at 5±3°C for 24 months from the date of manufacture met both BET and sterility acceptance criteria (see Table 17). Batch BL.14.0901 / R / 17 / 021 F DP BCA101 DP was not tested for BET and sterility.

[0285] Collection capacity Due to dead volume present in the vial and stopper that come into contact with the product during storage, it is not possible to completely extract 100% of the product from the vial. This was determined to be approximately 0.15–0.2 ml, and considering the filling accuracy of the machine, the filling volume of BCA101 DP was set to 10.0 mL or more (NLT). The sampling volume was therefore set to NLT 10.0 ml. As shown in Figure 43 (and Table 17), the BS18002245 BCA101 DP batch met the specification limits for 24 months from the date of manufacture. The BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was not tested for sampling volume.

[0286] Sealing integrity test The container closure integrity test (CCIT) complements the sterility test and is performed to ensure microbiological integrity (sterility) from storage and transport to the end of the DP shelf life. The acceptance criterion for the CCIT was set as "all tested vials contain no trace of any colored solution." The BS18002245 BCA101 DP batch met the specifications for 24 months from the date of manufacture (Table 17). The BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was not tested by CCIT.

[0287] Purity measured by SEC-HPLC SEC-HPLC provides information on the monomer content and associated HMWP of the product. Acceptance criteria for BCA101 DS were set as follows: monomer % NLT 93.00%, HMWP % NMT 3.00%, and LMWP as reported results. As shown in Figure 44 (HMWP%), Figure 45 (monomer %), and Figure 46 (LMWP%), the SEC-HPLC purity results met the specification limits for both BCA101 DP batches up to 24 months from the filling / manufacturing date.

[0288] Purification by RP-HPLC RP-HPLC provides purity information regarding the hydrophobic variants of the product. Acceptable criteria for these BCA101 DS samples were set as follows: main peak NLT 70.0%, total post-peak NMT 24%, and total pre-peak NMT 6%. As shown in Figure 47 (total pre-peak), Figure 48 (total post-peak), and Figure 49 (main peak %), the RP-HPLC purity results met the specification limits for the tested BS18002245 BCA101 DS batch up to 24 months from the manufacturing date.

[0289] Except for the T3M time point for the BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch, the hydrophobic variants of the product-related DP batches met the specification limits for both BCA101 DP batches at 24 months from the manufacturing date. The out-of-specification result observed at T3M alone for the BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was attributed to analytical assay deviation, as the stability trend was observed to meet acceptable criteria up to 24 months from the subsequent time point. This behavior was not observed in other analytical tests, and the stability trend of the BS18002245 BCA101 DP batch up to 24 months from the manufacturing date was within the specified limits.

[0290] Bifunctional ELISA The co-binding efficacy of BCA101 to EGFR and TGFβ1 ligands was determined using a bifunctional ELISA. Briefly, plates coated with recombinant hEGFR Fc were blocked and then incubated with BCA101 for approximately 1 hour, followed by incubation with recombinant hTGFβ1. hTGFβ1 bound to the hTGFβRII ECD portion of BCA101 was then detected with a biotinylated anti-hTGFβ1 antibody, followed by streptavidin-HRP. This ensured that a signal would be obtained only if both binding arms were intact. Assay acceptance criteria were set at an average relative potency of 0.80–1.25 compared to a reference standard.

[0291] As shown in Figure 50 (and Table 16), the relative potency of the BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch remained well within assay acceptance limits up to 24 months from the filling / manufacturing date, which was the last time point tested. Similarly, the results for the BS18002245 BCA101 DP batch remained well within assay acceptance limits up to 24 months from the filling / manufacturing date, which was the last time point tested (see Figure 50 and Table 17).

[0292] Growth inhibition (IOP) assay The ability of BCA101 DP to inhibit FaDu cancer cell growth by binding to its target EGFR was determined using an Inhibition on Growth (IOP) assay. This assay provides a method for determining the number of viable cells in a culture by quantifying the amount of ATP present. Readings were based on the luminescence of luciferin catalyzed by luciferase in the presence of Mg2+, and the ATP released by viable cells. The assay tolerance was set at an average relative potency of 0.80–1.25 compared to a reference standard.

[0293] As shown in Figure 51 (and Table 17), batch BS18002245 BCA101 DP met IOP standards for up to 24 months from the filling / manufacturing date. BL.14.0901 / R / 17 / 021 F DP BCA101 DP was not tested by IOP.

[0294] conclusion The data described above shows that multiple batches of BCA101 drug product stored at 5±3°C met acceptable criteria for up to 24 months from the filling / manufacturing date. Based on stability trend analysis, no significant changes were observed in pH, osmolality by weight, number of microscopic particles per container, SEC-HPLC, protein content, RP-HPLC, or functionality. Data obtained from R&D (BL.14.0901 / R / 17 / 021 F DP) and development GMP batch (BS18002245) demonstrate the physicochemical and functional stability of BCA101 DP over 24 months from the filling / manufacturing date when stored at 5±3°C in USP Type 1 glass vials. The BS18002245 BCA101 DP batch further met the BET and sterility specification limits for 24 months from the filling / manufacturing date, which was the longest time point tested.

[0295] [Example 4] Physicochemical and biological characterization of BCA101 DS The objective of this study was to further characterize and confirm the physicochemical and biological properties of two batches of BCA101 DS: 1) BL.14.0901 / R / 17 / 021 / F containing 25.58 mg / ml of BCA101, 0.02 wt / vol% of polysorbate 20, 8.0 wt / vol% of sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0). DS preclinical R&D batch and internal reference standard, as well as GF19000040 of GMP batch containing 26.60 mg / ml of BCA101, 0.02 wt / vol% of polysorbate 20, 8.0 wt / vol% of sucrose, and 10 mM citrate phosphate (0.573 mg / ml citrate monohydrate, 1.294 mg / mL disodium hydrogen phosphate dihydrate) buffer (pH 6.0). Table 18 provides an overview of the analytical tools used to evaluate the physicochemical and biological quality attributes of BCA101 in this example.

[0296] [Table 18]

[0297] The data from this embodiment demonstrate the equivalence of BCA101 GMP DS batch GF19000040 to the internal reference standard batch BL.14.0901 / R / 17 / 021 / F DS, indicating that both batches meet the established quality attribute specifications. A summary of the physicochemical and biological characterizations performed in this embodiment is provided below and in Table 19, and additional detailed descriptions of each analysis performed are provided below.

[0298] Primary structure: The intact molecular weight of batch GF19000040 was found to be equivalent to that of batch BL.14.0901 / R / 17 / 021 / F DS, and both were found to be within the quality attribute specifications (Figure 53 and Table 23). The intact molecular weight was found to be higher than the predicted theoretical molecular weight due to extensive glycosylation. The peptide sequences of batch GF19000040 and batch BL.14.0901 / R / 17 / 021 / F DS were consistent, including the N-terminus, C-terminus, and linker sequences, and were confirmed using the multi-enzyme PMF method (Figures 54-59).

[0299] Second- and even higher-order structures: The far and near UV CD profiles of batch GF19000040 were found to be equivalent to those of batch BL.14.0901 / R / 17 / 021 / F DS, and both were found to be within the quality attribute specifications (Figures 60-61). A negative signature peak was observed around 215 nm, indicating a characteristic β-sheet protein. All disulfide linkages present in batch GF19000040 were identified and confirmed to align with batch BL.14.0901 / R / 17 / 021 / F DS (Tables 24-26).

[0300] Glycosylation: The N-glycan profile of batch GF19000040 was determined to be equivalent to that of batch BL.14.0901 / R / 17 / 021 / F DS and within the range of quality attribute specifications (Tables 27-28 and Figures 63-64). The relative abundance of major glycoforms in batch GF19000040 was determined to be equivalent to that in batch BL.14.0901 / R / 17 / 021 / F DS. The sialic acid content in batch GF19000040 was estimated to be 8.8 moles per mole of protein, while that of batch BL.14.0901 / R / 17 / 021 / F DS was estimated to be 12.2 moles per mole of protein. This difference in average sialic acid content was determined to be within the range of methodological variability and to have little effect on biological function.

[0301] Biological activity: The biological activity of the BCA101 batch was evaluated in terms of its ability to simultaneously bind to its targets (EGFR and TGFβ1), inhibit alloreceptor-mediated signaling, and trigger ADCC via its Fc domain. Overall data from the biological assays showed that the biological activity of both the GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches was equivalent and within the quality attribute specifications (Tables 30-32).

[0302] Product-related variants: Size Variants - Percentages of monomers, HMWPs, and LMWP species were equivalent when analyzed by SEC-HPLC in both GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches and were within the quality attribute specifications (Table 20). Relative percentages of fragments quantified using nrCE-SDS and rCE-SDS were also equivalent and within specifications between GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches (Table 21). Charge Variants - Charge variants obtained after integration of iCE analysis were referred to as Region 1, Region 2, and Region 3. The profiles corresponded to each other, and the values ​​for GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches were equivalent and within the quality attribute specifications (Table 22). The RP profiles, showing the hydrophobic variant main, all pre-peaks, and all post-peaks, as well as the corresponding relative area percentages, were equivalent between GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches and were within the quality attribute specifications (Table 29).

[0303] [Table 19-1]

[0304] [Table 19-2]

[0305] Purity of BCA101 DS by SEC-HPLC Product-related size variant impurities include high molecular weight protein (HMWP) species, low molecular weight protein (LMWP) species, and fragments. HMWP species are formed as a result of the association of two or more monomer molecules. The primary analytical method for the separation and estimation of HMWPs is size exclusion chromatography-HPLC (SEC-HPLC), which was used here. As shown in Table 20, the SEC-HPLC profiles of the first batch (GF19000040) and the second batch BL.14.0901 / R / 17 / 021 / F DS (internal reference standard) of BCA101 DS were visually similar (chromatograms are not shown), and the relative percentages of monomers, HMWPs, and LMWPs were determined to be equivalent (see Table 20). In addition, the monomer, HMWP, and LMWP levels of both batches of BCA101 DS are within the specified ranges of 3.0% NMT for HMWP and 7.0% for LMWP, respectively (see Table 20).

[0306] [Table 20]

[0307] Purity of BCA101 DS using nrCE-SDS and rCE-SDS Capillary electrophoresis (CE) is an automated and instrumental version of conventional slab gel electrophoresis (SDS-PAGE) that utilizes narrow-bore (20-200 μm inner diameter) capillaries to achieve highly efficient separation of both large and small molecules. These separations are facilitated by the use of high voltage, which can generate electroosmotic and electrophoretic flows of buffer solutions and ionic species within the capillary, respectively. CE-SDS (reduced and non-reduced) is used for the quantitative analysis of heterogeneity based on purity and size of therapeutic products. Both BCA101 DS batches, GF19000040 and BL.14.0901 / R / 17 / 021 / F DS, were analyzed quantitatively for product-related fragments using non-reduced and reduced CE-SDS. The (non-reduced (nr) and reduced (r)) CE-SDS electrophoresis maps showed similarity across the two batches (chromatograms are not shown), both within the quality attribute specifications (Table 21). The main peak group in nrCE-SDS was observed to be extensive and bifurcated, which may result from heterogeneous three-dimensional structures in the denatured state, due to a) incomplete denaturation of large proteins and b) heterogeneity in glycoforms. nrCE-SDS analysis of batch GF19000040 and batch BL.14.0901 / R / 17 / 021 / F DS showed corrected area percentages of the main peak group as 94.3% and 94.4%, respectively. The rCE-SDS analysis showed that the ratio of the total most abundant species (peak 1 + peak 2 + peak 3) in batch GF19000040 and IRS was 97.5% and 97.8%, respectively. Therefore, the fragment ratios were similar across the two batches tested and were within the specifications (Table 21).

[0308] [Table 21]

[0309] Purity of BCA101 DS by iCE Image capillary electrophoresis (iCE) is a widely used analytical tool for the separation and quantification of product-related charge variants in biopharmaceuticals. This technique utilizes the principle of protein charge separation in a pH gradient gel matrix under applied current. Based on net charge, proteins move within the gel matrix until equilibrium is achieved between pH units and molecular isoelectric point (pI). Charge variant profiles of both BCA101 DS batches were evaluated using the iCE technique. Due to the heterogeneity of BCA101 resulting from severe sialylation, the charge variant profiles exhibit multiple peaks with a lack of baseline separation. Therefore, for ease of comparison, the peaks were clustered into regions R1, R2, and R3, as shown in Table 52. The relative proportions of regions R1, R2, and R3 were estimated from the area under the relative curve of each cluster. As shown in Table 22, the two batches were observed to be equivalent within method variability and within specifications.

[0310] [Table 22]

[0311] Intact Mass Spectrometry Intact mass spectrometry of BCA101 was performed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS is a routine, high-speed quantitative tool performed by applying a laser beam to a sample embedded in a rapidly ionizing matrix. The matrix absorbs ultraviolet light from the laser (a nitrogen laser with a wavelength of 337 nm) and converts it into thermal energy. A small portion of the matrix is ​​rapidly heated and vaporizes along with the sample, and the resulting spectrum of ionization yields monovalent ions. Based on the cluster size distribution of the mass spectrum, the software compartmentalizes the mean molecular mass of the analyzed sample. Intact mass spectra of batch GF19000040 and BL.14.0901 / R / 17 / 021 / F DS are shown in Figure 53 and Table 23.

[0312] As mentioned above, the theoretical molecular mass of BCA101 based on its amino acid sequence is 178105 kDa. As shown in Table 23, the observed molecular mass of BCA101 in batches GF1900040 and BL.14.0901 / R / 17 / 021 / F DS was approximately 192 kDa, which is higher than the theoretical molecular mass due to glycosylation. Due to heterogeneity in N-linked glycosylation in BCA101, the mass spectrum shows a broad molecular weight distribution in the range of 180 kDa to 210 kDa.

[0313] [Table 23]

[0314] Peptide mass fingerprinting Reduced peptide mass fingerprinting (PMF) provides detailed information about proteins, including determination of the primary sequence, N and C-terminal sequences, and the site and type of post-translational modifications. This approach involves subjecting the molecule to a specific enzymatic digestion procedure, followed by MS and / or MS. 2 The peptides are subjected to chromatographic separation before analysis. Tandem MS or MS. 2 The approach facilitates the evaluation of primary structure in terms of linker confirmation and N-terminal and C-terminal sequencing. N-terminal and C-terminal sequencing provide important datasets for confirming the start and end of the target protein sequence. PMF analysis is widely used in the pharmaceutical industry to generate a unique fingerprint of the target molecule and aid in identifying complete protein sequence coverage. PMF profile overlays for BCA101 batch GF19000040 and BCA101 batch BL.14.0901 / R / 17 / 021 / F DS were constructed (Figure 54). For LC-TGFβRII, 100% sequence coverage could be observed using trypsin and Glu-C, while for HC, four enzymes, trypsin, Glu-C, Asp-N, and LysC, were used for 100% sequence coverage. The heavy and light chain-linker-TGFβRII ECD fragments produced by multi-enzyme digestion were evaluated along with their theoretical and observed masses and their respective retention times.

[0315] As shown in Figure 54, the UV chromatograms of the GF19000040 batch and the BL.14.0901 / R / 17 / 021 / FDS batch are equivalent to each other and within the specifications. Linker MS 2 It was found to be intact. Using multiple enzymes (Glu-C, AspN, LysC), no free-end light chains were found, and therefore, it can be concluded that the fusion is intact in both batches.

[0316] N-terminal and C-terminal sequencing The start and end of the BCA101 protein sequence were confirmed by sequencing experiments of the N-terminus and C-terminus. PMF was obtained using MS and MS. 2 MS is a powerful method that uses data to provide N-terminal and C-terminal sequences and compares experimental spectral data with the masses of in silico-produced trypsin-digested LC and HC fragments. 2 The b and y daughter ion series of the N-terminal and C-terminal sequences of HC, LC, and the linker obtained from the spectra are shown in Figures 55 to 59.

[0317] From the data shown in Figures 55 to 59, the N-terminal and C-terminal sequences of the heavy and light chain TGFβRII were identified as follows: N-terminal HC:pyroQVQLK (SEQ ID NO: 35), C-terminal HC:SLSLSPG, N-terminal LC-TGFβRII:DILLTQSPVILSVSPGER (SEQ ID NO: 36), and LC-linker-TGFβRII:GECGGGGSGGGGSGGGGSTIPPHVQK (SEQ ID NO: 37). The C-terminus of TGFβRII ECD was found to have low strength and high molecular weight due to MS 2 Although this was not selected, the supported charge state was visible (MS data is provided above).

[0318] As shown in Figure 54, the UV chromatograms of the trypsin peptide maps of GF19000040 and BL.14.0901 / R / 17 / 021 / F DS correspond to each other, and the sequences were observed to be identical to the theoretical sequences. The first amino acid of the heavy chain at the N-terminus is "Gln," i.e., "Q," which was observed as pyro-Q in both batches analyzed. Pyro-glutamic acid (Q) is the result of spontaneous cyclization of glutamine. The heavy chain C-terminal amino acid sequence ends with "PG" and does not indicate the presence of lysine as "PGK." The N-terminus of the light chain is intact and shows no modifications in the FmAb2 batch. The C-terminus of the light chain is fused to TGFβRII ECD. The C-terminal sequence of TGFβRII ECD was determined to be intact and consistent with the available theoretical sequence.

[0319] Circular dichroism (CD) Circular dichroism is a form of absorption spectroscopy that measures the difference in absorption of right- and left-circularly polarized light from a substance. The secondary structure of a protein can be determined by CD spectroscopy in the "far-UV" spectral region (200–260 nm). At these wavelengths, a signal is produced when the chromogen is a peptide bond and located in a regularly folded environment. α-helical, β-sheet, and random coil structures yield CD spectra of characteristic shapes and sizes, respectively.

[0320] The CD spectrum of a protein in the near-UV spectral region (260–350 nm) may be sensitive to certain aspects of its tertiary structure. At these wavelengths, the chromogens are aromatic amino acids and disulfide bonds, and the resulting CD signals are sensitive to the overall tertiary structure of the protein. Signals in the 250–270 nm region are attributed to phenylalanine residues, signals in the 270–290 nm region to tyrosine, and signals in the 280–300 nm region to tryptophan. Because tertiary structure is protein-specific, it does not have a standard profile.

[0321] Figures 60 and 61 provide the far-UV and near-UV CD spectra of the BCA101 DS GF19000040 batch and the BCA101 BR.14.09015 / R / 16 / 021 DS batch, respectively. As shown in Figures 60-61, the profiles correspond to each other, with the BCA101 BR.14.09015 / R / 16 / 021 DS batch showing a minimum wavelength at 216.2 nm and the BCA101 DS GF19000040 batch showing a minimum at 215.4 nm, exhibiting similar secondary and tertiary structures. Negative signature peaks in the far-UV spectra of both batches were observed around 215 nm, indicating a characteristic β-sheet structure.

[0322] Non-reducing disulfide bridges The heavy chain, light chain, and disulfide linkages between the heavy and light chains of the BCA101 antibody skeleton determine its structure, stability, and biological function. The BCA101 antibody skeleton belongs to the IgG1 class and has 16 disulfide bonds: four interchain disulfide bonds in the hinge region and 12 intrachain bonds associated with different domains. Of the four interchain disulfides, two link the heavy chains together, and the other two connect the light and heavy chains. The C-terminal Cys 214 of the light chain forms a disulfide bond with Cys 222 of the heavy chain, which connects the light and heavy chains. Cys 228 and Cys 231 in each of the heavy chains link together to form two parallel interchain disulfide bonds between the two heavy chains. The 4-polypeptide chain (two heavy chains and two light chains) is linked by four interchain disulfide bonds to form a tetramer, which plays a crucial role in the antibody's skeletal structure and function. Disulfide scrambling or incomplete disulfide bond formation can lead to loss of function. In addition, the TGFβRII-ECD domain of BCA101 is rich in Cys residues, which are linked to form six intrachain disulfide crosslinks, which play a crucial role in the structure and function of the receptor-binding domain. Disulfide linkage was analyzed to establish the presence of correct linkages to ensure the function and quality of the drug.

[0323] Mass spectrometry (ESI-MS) tools were used to characterize the disulfide bond structure, which involves enzymatic cleavage of the protein, where peptides derived from proteolysis contain cysteine ​​residues to determine the presence and location of the disulfide bond. The peptide mixture was directly analyzed by mass spectrometry peptide mapping. Using molecular mass spectrometry, disulfide-bonded dipeptides were identified by assigning the observed ion signals to the mass calculated from the corresponding primary amino acid sequence. The disulfide bond structure of BCA101 was characterized using non-reducing peptide mapping. Generally, this method involves characterizing disulfide bonds without reduction through collision-induced dissociation (CID), which ensures selective cleavage of the protein and the generation of peptide mass fingerprinting under non-reducing conditions. The disulfide bond ligation observed in the antibody backbone and TGFβRII-ECD domain of BCA101 is presented in Table 24.

[0324] [Table 24]

[0325] Among the disulfide linkages in TGFβRII ECD, two trypsin peptides showed the presence of multiple disulfide bonds (peaks 10 and 11 in Table 25). Further characterization studies were performed using multiple enzyme digestion and MS to identify the specific linkages. 2 I used confirmation. Also, MS 2 We used this to verify the Cys258-Cys261 connection.

[0326] [Table 25]

[0327] Table 26 shows the predicted mass of peptides obtained by multi-enzyme digestion, along with their retention times (RT).

[0328] [Table 26]

[0329] As observed in Figure 62, the non-reducing PMF UV chromatogram of the BCA101 DS GF19000040 batch is equivalent to that of the BCA101 BL.14.0901 / R / 17 / 021 / F DS batch and is within the specification range. The masses of the disulfide-linked peptides after proteolytic digestion with multiple enzymes for both batches are shown in Tables 25 and 26, and were found to be equivalent across batches and to have the corresponding theoretical masses. Of the six disulfide linkages in the TGFβRII ECD, three, Cys258-Cys261 (AspN), Cys268-Cys274 (AspN / PNGaseF), and Cys291-Cys308 (Trp / GluC), were 2 confirmed by MS analysis. Furthermore, in one of the peptides generated by AspN digestion (...NCSIT....TVCH), Cys291-Cys308 had already been confirmed by GluC digestion peptide MS 2 analysis, so the only possibility for the other disulfide bond was between Cys278-Cys284, which was confirmed as the fourth disulfide linkage. The masses of the corresponding peptides with disulfide bonds were confirmed and were equivalent across the two batches. Therefore, the disulfide-linked peptides in the TGFβRII ECD were confirmed by multiple enzyme digestion and MS 2 confirmation.

[0330] N-glycan analysis using normal-phase liquid chromatography (NP-HPLC) and ESI-MS The heterogeneity of biotherapeutics expressed in mammalian cells is mostly due to post-translational modifications, such as glycosylation in conserved regions of the protein. N-linked glycosylation in monoclonal antibodies plays an important role in ligand / antigen binding and its functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and clearance rate.

[0331] To determine the N-linked glycosylation of BCA101, samples were denatured with SDS, deglycosylated using PNGaseF, and the released glycans were labeled with 2-aminoanthranilic acid (2-AA). These glycans were then separated and quantified by FLD-NP-HPLC. The mass of each glycan species was determined using LC-ESI-MS.

[0332] Table 27 below provides the relative abundances of observed glycan species. The mass identification of glycan species was obtained using LC-ESI-MS. 2 This was confirmed by the following: The 2-AA labeled N-glycan NP-HPLC chromatograms and relative abundance percentages (Table 27) for batches BCA101 DS GF19000040 and BCA101 DS BL.14.0901 / R / 17 / 021 / F DS, shown in Figure 63, are equivalent and within specifications.

[0333] [Table 27]

[0334] Sialic acid content Sialic acid exists in two forms: N-glycosylneuraminic acid (NGNA) and N-acetylneuraminic acid (NANA). Human glycosylated proteins contain the NANA form of sialic acid. Among the various monosaccharides present in Fc glycans, terminal sialic acids are of particular interest because they play different roles in monoclonal antibody function.

[0335] Sialic acid acts as a cap, hiding the second-to-last galactose residue recognized by the hepatic asialoglycoprotein receptor; therefore, the half-lives of some glycoproteins can be enhanced by sialylation. Consequently, a better understanding and monitoring of sialyzed glycans is needed from the perspective of obtaining safe and reliable monoclonal antibodies for the application of various biotherapeutic drugs.

[0336] In addition to affecting the biological and physicochemical properties of biopharmaceuticals, the sialic acid moiety of protein therapies plays a major role in serum half-life because the galactose exposed as a glycoprotein is endocytotic by the hepatic asialogalactose receptor via receptor-mediated endocytosis. Sialic acid release is via acid hydrolysis of monoclonal antibodies, followed by cleanup to remove the monoclonal antibodies and labeling of the sialic acid species with a fluorescent tag using an OPD. The tagged samples are injected into a reversed-phase high-performance liquid chromatography (RP-HPLC) column for analysis. The linear dynamic calibration range of the assay is determined by running a set of NANA standards at different concentrations.

[0337] The data overlay shown in Figure 64 shows that the peak corresponding to NGNA is absent in BCA101 DS based on 25 and 300 pmol NGNA standard samples, and only the peak corresponding to NANA (based on the peak observed in the NANA standard sample) is shown. Therefore, the data indicates the presence of NANA in BCA101, and that NGNA was not detected up to 20 pmol. As shown in Table 28, the mean (n=4) sialic acid content in BCA101 DS (GF19000040) is estimated to be 8.8 mol per mole of protein, and in BCA101 DS (BL.14.0901 / R / 17 / 021 / F DS) it is estimated to be 12.2 mol per mole of protein. This difference in mean sialic acid content is within the range of method variability and has little effect on the biological function between batches, as shown in the functional assays presented below.

[0338] [Table 28]

[0339] Product-related materials - RP - HPLC Reverse-phase HPLC (RP-HPLC) is used to monitor product-related hydrophobic variants, including post-translational modifications, oxidized proteins, clip variants or fragments, and N-terminal cyclizations. Based on hydrophobicity, proteins are separated into columns, with less hydrophobic proteins eluting earlier than more hydrophobic variants.

[0340] Product-related hydrophobic variants in the BCA101 batch were classified into main peaks, pre-main peaks (grouped together as all pre-peaks), and post-main peaks (grouped together as all post-peaks). The RP profiles and relative ratios of the main peaks, pre-peaks, and post-peaks were observed to be similar across two BCA101 DS batches (GF19000140 and BL.14.0901 / R / 17 / 021 / F DS) (Table 29). The total main peak content was observed to be 82.2% (GF19000140) and 81.3% (BL.14.0901 / R / 17 / 021 / F DS), both within the specification range of 70% or more (NLT). The total pre-peak percentage was observed to be 4.9% (GF19000140) and 4.8% (BL.14.0901 / R / 17 / 021 / F DS), both within the specification range of 6.0% or less (NMT). The total post-peak content was observed to be 12.9% (GF19000140) and 13.9% (BL.14.0901 / R / 17 / 021 / F DS), both within the specification range of 24.0% or less (NMT).

[0341] [Table 29]

[0342] Inhibition of growth (IOP) BCA101 binds to EGFR on FaDu cancer cells. Variations in drug concentration allow for dose-dependent inhibition of cancer cell proliferation in this assay. The cell Titer-Glo® 2.0 assay provides a homogeneous method for determining the number of viable cells in a culture by quantifying the amount of ATP present, which indicates the presence of metabolically active cells. Readings are based on the luminescence of luciferin catalyzed by luciferase in the presence of Mg2+, and the ATP released by viable cells.

[0343] The GF19000040 batch was analyzed in three independent experiments using the BL.14.0901 / R / 17 / 021 / F DS batch as the standard. The average relative potency from the three experiments is shown in Table 30 below.

[0344] As shown in Table 30, batch GF19000040 exhibited an average relative potency of 0.99, which falls within the assay acceptance range of 0.8–1.25. The results indicate that GF19000040 and BL.14.0901 / R / 17 / 021 / F DS exhibit similar potency in inhibiting FaDu cell proliferation.

[0345] [Table 30]

[0346] Bifunctional ELISA As described above, a bifunctional ELISA assay was used to determine the binding efficacy of the fusion portion of BCA101 to its target protein, thereby determining that both portions of BCA101 are simultaneously functional. FmAb2 has a TGFβRII ECD fused to an anti-EGFR monoclonal antibody at the C-terminus of its light chain. The TGFβRII ECD portion primarily binds to TGFβ1, while the anti-EGFR portion binds to EGFR. Individual target-binding ELISAs can only independently determine the binding affinity of each portion and cannot determine bifunctionality.

[0347] The bifunctional activity of batch GF19000040 was evaluated using BL.14.0901 / R / 17 / 021 / F DS as the standard. As shown in Table 31, GF19000040 showed a relative potency value of 0.93, which is well within the acceptable range of 0.8 to 1.25. The results further indicate that GF19000040 exhibits similar potency to BL.14.0901 / R / 17 / 021 / F DS in terms of bifunctional activity.

[0348] [Table 31]

[0349] Antibody-dependent cytotoxicity (ADCC): The ADCC assay evaluates the Fc function of BCA101. The ADCC reporter bioassay is a bioluminescent reporter assay that uses an alternative early-stage readout of the ADCC mechanism of action by activating gene transcription through NFAT (nuclear factor of activated T cells) in effector cells. The ADCC reporter bioassay is performed using an ADCC bioassay effector cell proliferation model (Promega, catalog no. G7102) that enables cell banking and proliferation. These cells are engineered Jurkat cells that stably express the FcγRIIIa receptor V158 (high affinity) variant. Biological activity in ADCC is quantified via luciferase produced as a result of NFAT pathway activation. Luciferase activity in effector cells is quantified by luminescence reading.

[0350] The ADCC activity of batch GF19000040 was evaluated using FmAb2 IRS as the standard. As shown in Table 32, batch GF19000040 showed a relative potency value of 1.23, which is within the acceptable range of 0.8 to 1.25, and also showed a CV% of 20 or less. The results indicate that batch GF19000040 showed similar potency in terms of ADCC activity compared to batch BL.14.0901 / R / 17 / 021 / F DS.

[0351] [Table 32]

[0352] TGFβ SMAD assay The TGFβ SMAD assay is a functional assay that determines the functional activity of the TGFβRII ECD arm of a fusion antibody. Briefly, the HEK293 cell line is manipulated to determine the activity of the TGFβ-SMAD signaling pathway. The cell line contains a firefly luciferase gene under the control of a SMAD-responsive element stably integrated into HEK293 cells. The TGFβ protein binds to receptors on the cell surface, initiating a signaling cascade that results in the phosphorylation and activation of SMAD2 and SMAD3, which then form a complex with SMAD4. The SMAD complex then translocates to the nucleus and binds to the SMAD-binding element (SBE) in the nucleus, resulting in the transcription and expression of the TGFβ SMAD-responsive gene. Stimulation with human TGFβ1 increases the luminescence signal in a dose-dependent manner, which is neutralized in the presence of BCA101.

[0353] As shown in Table 33, batch GF19000040 was evaluated using BL.14.0901 / R / 17 / 021 / F DS as the standard. Batch GF19000040 showed a relative potency of 1.10, which is well within the acceptable range of 0.80 to 1.25, and also showed a CV% of 20 or less. This further establishes that batch GF19000040 is equivalent to batch BL.14.0901 / R / 17 / 021 / F DS.

[0354] [Table 33]

[0355] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0356] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in all their entirety for any purpose to the same extent that each individual reference (e.g., publication, patent, or patent application) is shown to be incorporated by reference in all its entirety for any specific and individual purpose.

[0357] Other embodiments are within the scope of the following claims. The present invention includes the following embodiments. <1> A liquid pharmaceutical composition, a. A fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to the human epidermal growth factor receptor (hEGFR), and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of the human transforming growth factor-beta receptor II (hTGFβRII), b. Buffer solutions present at concentrations of 5 mM to 30 mM, c.4 A tension adjusting agent present at a concentration of 10% by weight / volume and Includes, The liquid pharmaceutical composition has a pH of 5.5 to 7.0. Liquid pharmaceutical composition. <2> The buffer solution is citrate phosphate buffer, citrate buffer, succinate buffer, or histidine buffer. <1> The liquid pharmaceutical composition described above. <3> The buffer solution is citrate phosphate buffer. <1> ~ <2> A liquid pharmaceutical composition as described in any of the following. <4> The buffer solution is present at concentrations of 5mM to 25mM, 5mM to 20mM, 5mM to 15mM, 5mM to 10mM, or 10mM to 30mM. <1> ~ <3> A liquid pharmaceutical composition as described in any of the following. <5> The buffer solution is present at a concentration of 5 mM to 15 mM. <1> ~ <4> A liquid pharmaceutical composition as described in any of the following. <6> The buffer solution is present at concentrations of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM. <1> ~ <5> A liquid pharmaceutical composition as described in any of the following. <7> The buffer solution is present at a concentration of 10 mM. <1> ~ <6> A liquid pharmaceutical composition as described in any of the following. <8> The buffer solution contains 10 mM citrate phosphate. <1> ~ <7> A liquid pharmaceutical composition as described in any of the following. <9> The tension adjusting agent is sucrose or trehalose. <1> ~ <8> A liquid pharmaceutical composition as described in any of the following. <10> The aforementioned tension adjusting agent is a sugar. <1> ~ <9> A liquid pharmaceutical composition as described in any of the following. <11> The aforementioned tension adjusting agent is a disaccharide. <1> ~ <10> A liquid pharmaceutical composition as described in any of the following. <12> The tension adjusting agent is sucrose. <1> ~ <11> A liquid pharmaceutical composition as described in any of the following. <13> The tension adjusting agent is present at concentrations of 5 wt / vol% to 10 wt / vol%, 6 wt / vol% to 10 wt / vol%, 7 wt / vol% to 10 wt / vol%, 8 wt / vol% to 10 wt / vol%, 5 wt / vol% to 9 wt / vol%, 5 wt / vol% to 8 wt / vol%, 6 wt / vol% to 9 wt / vol%, 6 wt / vol% to 8 wt / vol%, 7 wt / vol% to 9 wt / vol%, or 7 wt / vol% to 8 wt / vol%. <1> ~ <12> A liquid pharmaceutical composition as described in any of the following. <14> The aforementioned tension adjusting agent is present at a concentration of 5% by weight / vol. to 8% by weight / vol. <1> ~ <13> A liquid pharmaceutical composition as described in any of the following. <15> The tension adjusting agent is present at concentrations of 5% by weight / vol, 6% by weight / vol, 7% by weight / vol, 8% by weight / vol, 9% by weight / vol, or 10% by weight / vol. <1> ~ <14> A liquid pharmaceutical composition as described in any of the following. <16> The aforementioned tension adjusting agent is present at a concentration of 8% by weight / volume. <1> ~ <15> A liquid pharmaceutical composition as described in any of the following. <17> The aforementioned tension adjusting agent is sucrose, and is present at a concentration of 8% by weight / volume. <1> ~ <16> A liquid pharmaceutical composition as described in any of the following. <18> Further containing surfactants, <1> ~ <17> A liquid pharmaceutical composition as described in any of the following. <19> The surfactant includes polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. <18> The liquid pharmaceutical composition described above. <20> The surfactant comprises polysorbate 20. <19> The liquid pharmaceutical composition described above. <21> The surfactant is present at a concentration of 0.005 to 0.1% by weight / volume. <18> ~ <20> A liquid pharmaceutical composition as described in any of the following. <22> The surfactant is present in concentrations of 0.01-0.1 wt / vol.%, 0.02-0.1 wt / vol.%, 0.01-0.9 wt / vol.%, 0.01-0.8 wt / vol.%, 0.01-0.7 wt / vol.%, 0.01-0.6 wt / vol.%, 0.01-0.5 wt / vol.%, 0.01-0.4 wt / vol.%, 0.01-0.3 wt / vol.%, 0.01-0.2 wt / vol.%, 0.01-0.1 wt / vol.%, 0.02-0.9 wt / vol.%, 0.02-0.8 wt / vol.%, 0.02-0.7 wt / vol.%, and 0.02-0.6 wt / vol. It exists at concentrations of %, 0.02-0.5 wt / vol.%, 0.02-0.4 wt / vol.%, 0.02-0.3 wt / vol.%, 0.02-0.2 wt / vol.%, 0.02-0.1 wt / vol.%, 0.005-0.9 wt / vol.%, 0.005-0.8 wt / vol.%, 0.005-0.7 wt / vol.%, 0.005-0.6 wt / vol.%, 0.005-0.5 wt / vol.%, 0.005-0.4 wt / vol.%, 0.005-0.3 wt / vol.%, 0.005-0.2 wt / vol.%, or 0.005-0.1 wt / vol. <18> ~ <21> A liquid pharmaceutical composition as described in any of the following. <23> The surfactant is present at concentrations of 0.01% by weight / volume, 0.02% by weight / volume, 0.03% by weight / volume, 0.04% by weight / volume, 0.05% by weight / volume, 0.06% by weight / volume, 0.07% by weight / volume, 0.08% by weight / volume, 0.09% by weight / volume, or 0.1% by weight / volume. <18> ~ <22> A liquid pharmaceutical composition as described in any of the following. <24> The surfactant is present at a concentration of 0.02% by weight / volume. <18> ~ <23> A liquid pharmaceutical composition as described in any of the following. <25> The surfactant is polysorbate 20, present at a concentration of 0.02% by weight / volume. <18> ~ <24> A liquid pharmaceutical composition as described in any of the following. <26> The liquid pharmaceutical composition has a pH of 5.5-7.0, 6.0-7.0, 5.5-6.5, 5.5-6.0, or 6.0-6.5. <1> ~ <25> A liquid pharmaceutical composition as described in any of the following. <27> The liquid pharmaceutical composition has a pH of 6.0 to 6.5. <1> ~ <26> A liquid pharmaceutical composition as described in any of the following. <28> The liquid pharmaceutical composition has a pH of 5.5, 6.0, 6.5, or 7.0. <1> ~ <27> A liquid pharmaceutical composition as described in any of the following. <29> The liquid pharmaceutical composition has a pH of 6.0. <1> ~ <28> A liquid pharmaceutical composition as described in any of the following. <30> The liquid pharmaceutical composition has a weight osmolality of 150 mOsmol / kg to 400 mOsmol / kg. <1> ~ <29> A liquid pharmaceutical composition as described in any of the following. <31> The liquid pharmaceutical composition has a gravimetric osmolality of 150 mOsmol / kg to 350 mOsmol / kg, 150 mOsmol / kg to 300 mOsmol / kg, 200 mOsmol / kg to 400 mOsmol / kg, 250 mOsmol / kg to 400 mOsmol / kg, 300 mOsmol / kg to 400 mOsmol / kg, 300 mOsmol / kg to 350 mOsmol / kg, 250 mOsmol / kg to 350 mOsmol / kg, or 250 mOsmol / kg to 300 mOsmol / kg. <1> ~ <30> A liquid pharmaceutical composition as described in any of the following. <32> The liquid pharmaceutical composition has a weight osmolality of 250 mOsmol / kg to 350 mOsmol / kg. <1> ~ <31> A liquid pharmaceutical composition as described in any of the following. <33> The liquid pharmaceutical composition has a weight-osmolality of 250 mOsmol / kg, 300 mOsmol / kg, or 300 mOsmol / kg. <1> ~ <32> A liquid pharmaceutical composition as described in any of the following. <34> The liquid pharmaceutical composition has a weight osmolality of 300 mOsmol / kg. <1> ~ <33> A liquid pharmaceutical composition as described in any of the following. <35> The liquid pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at -20°C. <1> ~ <34> A liquid pharmaceutical composition as described in any of the following. <36> The liquid pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at 2-8°C. <1> ~ <35> A liquid pharmaceutical composition as described in any of the following. <37> The concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at -20°C. <1> ~ <36> A liquid pharmaceutical composition as described in any of the following. <38> The concentration of the fusion protein in the liquid pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at 2-8°C. <1> ~ <37> A liquid pharmaceutical composition as described in any of the following. <39> The concentration of the fusion protein in the liquid pharmaceutical composition shall not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -20°C for 12 months, 18 months, or 24 months. <1> ~ <38> A liquid pharmaceutical composition as described in any of the following. <40> The concentration of the fusion protein in the liquid pharmaceutical composition does not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at 2-8°C for 12 months, 18 months, or 24 months. <1> ~ <39> A liquid pharmaceutical composition as described in any of the following. <41> The liquid pharmaceutical composition is stable after one, two, three, four, or five freeze-thaw cycles. <1> ~ <40> A liquid pharmaceutical composition as described in any of the following. <42> The fusion protein retains its bifunctional activity, as measured by a bifunctional enzyme-linked immunosorbent assay (ELISA), for at least 12 months, 18 months, or 24 months when stored at -20°C. <1> ~ <41> A liquid pharmaceutical composition as described in any of the following. <43> The fusion protein retains its bifunctional activity, as measured by bifunctional ELISA, for at least 12 months, 18 months, or 24 months when stored at 2-8°C. <1> ~ <42> A liquid pharmaceutical composition as described in any of the following. <44> The liquid pharmaceutical composition contains the fusion protein in aggregate form in an amount of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. <1> ~ <43> A liquid pharmaceutical composition as described in any of the following. <45> The liquid pharmaceutical composition After storage at -20°C or 2-8°C for 12 months, 18 months, or 24 months, compared to the control formulation, a. Increased shelf life, b. Increased temperature stability, c. reduced aggregate formation, d. Increased chemical stability, and / or e. Reduced fragment formation, f. Decreased viscosity Having at least one characteristic selected from the group consisting of, <1> ~ <44> A liquid pharmaceutical composition as described in any of the following. <46> The liquid pharmaceutical composition After storage at -20°C or 2-8°C for 12, 18, or 24 months, compared to the reference formulation, a. Percentage reduction of aggregates, as measured by size exclusion chromatography (SEC). b. High monomer percentage, and / or as measured by SEC. c. Low turbidity values ​​in turbidimetric units (NTU), Having at least one characteristic selected from the group consisting of, <1> ~ <45> A liquid pharmaceutical composition as described in any of the following. <47> The fusion protein is present at concentrations of 5-50 mg / ml, 5-40 mg / ml, 5-30 mg / ml, 5-25 mg / ml, 10-50 mg / ml, 20-50 mg / ml, 25-50 mg / ml, 20-50 mg / ml, 20-40 mg / ml, 20-30 mg / ml, 25-50 mg / ml, 25-40 mg / ml, or 25-30 mg / ml. <1> ~ <46> A liquid pharmaceutical composition as described in any of the following. <48> The aforementioned fusion protein is present at a concentration of 20-30 mg / ml. <1> ~ <47> A liquid pharmaceutical composition as described in any of the following. <49> The fusion protein is present at concentrations of 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. <1> ~ <48> A liquid pharmaceutical composition as described in any of the following. <50> The aforementioned fusion protein is present at a concentration of 25 mg / ml. <1> ~ <49> A liquid pharmaceutical composition as described in any of the following. <51> The targeting portion that specifically binds to hEGFR includes an antibody or its functional fragment or functional variant. <1> ~ <50> A liquid pharmaceutical composition as described in any of the following. <52> The antibody, or its functional fragment or functional variant, that specifically binds to hEGFR is a full-length antibody, a single-strand variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, or F(v). <51> The liquid pharmaceutical composition described above. <53> The antibody, or its functional fragment or functional variant, specifically binds to hEGFR and includes VH containing VH CDR1, VH CDR2, and VH CDR3. a. VH CDR1 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, b. VH CDR2 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 2. c.VH CDR3 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3. <51> or <52> The liquid pharmaceutical composition described above. <54> The antibody, or its functional fragment or functional variant, specifically binds to hEGFR and includes VLs containing VL CDR1, VL CDR2, and VL CDR3. a. VL CDR1 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. b. VL CDR2 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. c.VL CDR3 contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6. <51> ~ <53> A liquid pharmaceutical composition as described in any of the following. <55> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, contains a VH having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. <51> ~ <54> A liquid pharmaceutical composition as described in any of the following. <56> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, includes a VL containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. <51> ~ <55> A liquid pharmaceutical composition as described in any of the following. <57> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, includes a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. <51> ~ <56> A liquid pharmaceutical composition as described in any of the following. <58> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, consists of a heavy chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. <51> ~ <57> A liquid pharmaceutical composition as described in any of the following. <59> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, contains a heavy chain consisting of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. <51> ~ <57> A liquid pharmaceutical composition as described in any of the following. <60> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, consists of a heavy chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. <51> ~ <57> A liquid pharmaceutical composition as described in any of the following. <61> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, includes a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. <51> ~ <60> A liquid pharmaceutical composition as described in any of the following. <62> The antibody, or its functional fragment or functional variant, which specifically binds to hEGFR, consists of a light chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. <51> ~ <61> A liquid pharmaceutical composition as described in any of the following. <63> The antibody, or its functional fragment or functional variant, that specifically binds to hEGFR, comprises cetuximab or panitumumab, or a functional fragment or functional variant of either of the aforementioned. <51> The liquid pharmaceutical composition described above. <64> The immunomodulatory portion includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. <1> ~ <63> A liquid pharmaceutical composition as described in any of the following. <65> The immunomodulatory portion consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. <1> ~ <64> A liquid pharmaceutical composition as described in any of the following. <66> The immunomodulatory portion is indirectly fused to the targeting portion. <1> ~ <65> A liquid pharmaceutical composition as described in any of the following. <67> The immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker. <66> The liquid pharmaceutical composition described above. <68> The immunomodulatory portion is indirectly fused to the targeting portion via a peptide linker of sufficient length so that the immunomodulatory portion and the targeting portion can bind to their respective targets simultaneously. <67> The liquid pharmaceutical composition described above. <69> The linker includes the amino acid sequence of SEQ ID NOs. 24, 25, 26, 27, or 28. <66> or <67> The liquid pharmaceutical composition described above. <70> The linker includes the amino acid sequence of SEQ ID NO: 24, <67> ~ <69> A liquid pharmaceutical composition as described in any of the following. <71> The linker consists of the amino acid sequence of Sequence ID No. 24, <67> ~ <69> A liquid pharmaceutical composition as described in any of the following. <72> The immunomodulatory portion is fused to the C-terminus of the targeting portion. <1> ~ <71> A liquid pharmaceutical composition as described in any of the following. <73> The immunomodulatory portion is fused to the N-terminus of the targeting portion. <1> ~ <71> A liquid pharmaceutical composition as described in any of the following. <74> The targeting portion is an antibody comprising a light chain and a heavy chain, and the immunomodulatory portion is fused to the C-terminus of the heavy chain of the targeting portion. <1> ~ <73> A liquid pharmaceutical composition as described in any of the following. <75> The targeting portion is an antibody comprising a light chain and a heavy chain, and the immunomodulatory portion is fused to the C-terminus of the light chain of the targeting portion. <1> ~ <74> A liquid pharmaceutical composition as described in any of the following. <76> An antibody that specifically binds to hEGFR, wherein the targeted portion comprises a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the heavy chain or the light chain via a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24, <1> ~ <75> A liquid pharmaceutical composition as described in any of the following. <77> An antibody that specifically binds to hEGFR, wherein the targeted portion comprises a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11, wherein the immunomodulatory portion contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the light chain via a linker, and the linker contains an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24, <1> ~ <76> A liquid pharmaceutical composition as described in any of the following. <78> The antibody comprises a heavy chain in which the targeted portion contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. <1> ~ <77> A liquid pharmaceutical composition as described in any of the following. <79> The liquid pharmaceutical composition is sterile. <1> ~ <78> A liquid pharmaceutical composition as described in any of the following. <80> A liquid pharmaceutical composition, a. A fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of hTGFβRII. b. 5 mM to 20 mM citrate phosphate buffer, c.6% by weight / volume to 10% by weight / volume sucrose Includes, The liquid pharmaceutical composition has a pH of 5.5 to 6.5. Liquid pharmaceutical composition. <81> The antibody comprises a heavy chain in which the targeted portion contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. <80> The liquid pharmaceutical composition described above. <82> The aforementioned fusion protein is present at a concentration of 25 mg / ml. <80> or <81> The liquid pharmaceutical composition described above. <83> Further containing 0.01-0.05 weight / volume percent of polysorbate 20, <80> ~ <82> A liquid pharmaceutical composition as described in any of the following. <84> A liquid pharmaceutical composition, a. A fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises the amino acid sequence of the extracellular domain of hTGFβRII, b. 10 mM citrate phosphate buffer, c.8 Sucrose by weight / volume Includes, The liquid pharmaceutical composition has a pH of 6.0 ± 0.3. Liquid pharmaceutical composition. <85> Further comprising 0.02 wt / volt% polysorbate 20, <84> The liquid pharmaceutical composition described above. <86> The liquid pharmaceutical composition according to <84 or 85, wherein the targeted portion comprises an antibody comprising a heavy chain containing an amino acid sequence identical to at least 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence identical to at least 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 29. <87> The aforementioned fusion protein is present at a concentration of 25 mg / ml. <84> ~ <86> A liquid pharmaceutical composition as described in any of the following. <88> A liquid pharmaceutical composition, a. A fusion protein comprising a 25 mg / mL fusion protein including a targeting portion and an immunomodulatory portion, wherein the targeting portion comprises an antibody comprising a heavy chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain containing an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29, b. 10 mM citrate phosphate buffer, c.8 Sucrose in weight / volume %, d.0.02 wt / volume% polysorbate 20 and Includes, The liquid pharmaceutical composition has a pH of 6.0 ± 0.3. Liquid pharmaceutical composition. <89> A method for treating human cancer in a subject having cancer, wherein the subject <1> ~ <86> A method comprising the step of administering a liquid pharmaceutical composition as described in any of the above. <90> The liquid pharmaceutical composition is administered in an amount effective to treat the cancer. <89> Methods used. <91> The fusion protein is administered to the human subjects in doses of 50 mg to 2000 mg. <89> or <90> Methods used. <92> The fusion protein is administered to the human subjects in doses of 50 mg, 60 mg, 64 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. <89> ~ <91> One of the methods described above. <93> The fusion protein is administered to the human subjects in doses of 64 mg, 240 mg, 800 mg, or 1600 mg. <89> ~ <92> One of the methods described above. <94> The fusion protein is administered to the human subjects every week, every two weeks, every three weeks, or every four weeks. <89> ~ <93> One of the methods described above. <95> The fusion protein is administered to the human subject every week. <94> Methods used. <96> The fusion protein is administered to the human subjects every three weeks. <94> Methods used. <97> The administration step includes intravenous injection of the liquid pharmaceutical composition. <89> ~ <96> One of the methods described above. <98> The aforementioned cancer is a solid tumor. <89> ~ <97> One of the methods described above. <99> The aforementioned cancer is metastatic, recurrent, refractory, or any combination thereof. <89> ~ <98> The method described in any one of the items. <100> The aforementioned cancer includes cancer cells containing genomic amplification of the EGFR gene, which can be detected, for example, by biopsy or fluorescence in situ hybridization. <89> ~ <99> One of the methods described above. <101> The aforementioned cancer includes cancer cells that have undergone genomic modification in the KRAS gene. <89> ~ <100> One of the methods described above. <102> The aforementioned modification in the KRAS gene is a G12D substitution. <101> Methods used. <103> The aforementioned modification in the KRAS gene is the G13D modification. <101> Methods used. <104> The aforementioned cancer is selected from the group consisting of eye, stomach, colon, rectum, colorectal, breast, anal, pancreatic, thyroid, liver, ovarian, lung, skin, brain, spinal cord, head, and neck cancers. <89> ~ <103> One of the methods described above. <105> The aforementioned cancer is lung cancer. <89> ~ <104> One of the methods described above. <106> The aforementioned cancer is squamous cell lung cancer (SqCLC). <105> Methods used. <107> The SqCLC includes cancer cells that do not express a detectable level of programmed death ligand 1, as measured by biopsy. <106> Methods used. <108> The aforementioned SqCLC includes cancer cells containing EGFR gene genomic amplification, which can be detected, for example, by biopsy or fluorescence in situ hybridization. <106> or <107> Methods used. <109> The aforementioned cancer is colorectal cancer. <89> ~ <104> One of the methods described above. <110> The aforementioned colorectal cancer is RAS wild-type microsatellite-stable colorectal cancer (RAS WT MSS CRC). <109> Methods used. <111> The aforementioned cancer is breast cancer. <89> ~ <104> One of the methods described above. <112> The aforementioned cancer is triple-negative breast cancer (TNBC). <111> Methods used. <113> The aforementioned cancer is spinal cord cancer. <89> ~ <104> One of the methods described above. <114> The aforementioned spinal cord cancer is a chordoma. <113> Methods used. <115> The aforementioned cancer is eye cancer. <89> ~ <104> One of the methods described above. <116> The aforementioned eye cancer is ocular melanoma. <115> Methods used. <117> The aforementioned cancer is brain cancer. <89> ~ <104> One of the methods described above. <118> The aforementioned brain cancer is glioblastoma. <117> Methods used. <119> The aforementioned cancer is ovarian cancer. <89> ~ <104> One of the methods described above. <120> The aforementioned ovarian cancer is an epithelial ovarian cancer. <119> Methods used. <121> The aforementioned cancer is liver cancer. <89> ~ <104> One of the methods described above. <122> The aforementioned liver cancer is hepatocellular carcinoma (HCC). <121> Methods used. <123> The aforementioned cancer is thyroid cancer. <89> ~ <104> One of the methods described above. <124> The aforementioned thyroid cancer is undifferentiated thyroid cancer (ATC). <123> Methods used. <125> The aforementioned cancer is pancreatic cancer. <89> ~ <104> One of the methods described above. <126> The aforementioned cancer is stomach cancer. <89> ~ <104> One of the methods described above. <127> The aforementioned cancer is head and neck cancer. <89> ~ <104> One of the methods described above. <128> The aforementioned cancer is head and neck squamous cell carcinoma (HNSCC). <127> Methods used. <129> The aforementioned cancer is anal cancer. <89> ~ <104> One of the methods described above. <130> The aforementioned cancer is squamous cell carcinoma (SCCAC) of the anal canal. <129> Methods used. <131> A method for producing a liquid pharmaceutical composition, a. A step of culturing mammalian cells in which one or more nucleic acids encoding a fusion protein comprising a targeting portion and an immunomodulatory portion are stably incorporated into their genome, in a cell culture medium such that the cells secrete the fusion protein into the cell culture medium, wherein (i) the targeting portion specifically binds to hEGFR, and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain of hTGFβRII. b. A step of purifying the fusion protein from the cell culture medium, c. <1> ~ <88> A step of preparing a pharmaceutical composition as described in any of the above. A method that includes this. < / l>

Claims

1. A pharmaceutical composition, a. A fusion protein comprising a targeting portion and an immunomodulatory portion, (i) The targeting portion is an antibody that specifically binds to the human epidermal growth factor receptor (hEGFR), the targeting portion comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 10 and a light chain containing the amino acid sequence of SEQ ID NO: 11, the immunomodulatory portion comprises the amino acid sequence of SEQ ID NO: 23, the N-terminus of the immunomodulatory portion is indirectly fused to the C-terminus of the light chain via a linker, the linker comprises the amino acid sequence of SEQ ID NO: 24, (ii) The immunomodulatory portion comprises the amino acid sequence of the extracellular domain of human transforming growth factor-beta receptor II (hTGFβRII), Fusion protein and, b. Citrate phosphate buffer present at concentrations of 5 mM to 30 mM, c. Sucrose present at concentrations of 4% by weight / volume to 10% by weight / volume, d. Surfactants and Includes, The pharmaceutical composition has a pH of 5.5 to 6.

5. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a liquid pharmaceutical composition.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a powdered pharmaceutical composition.

4. The pharmaceutical composition according to claim 1, wherein the buffer solution is present in a concentration of 5 mM to 15 mM.

5. The pharmaceutical composition according to claim 1, wherein the buffer solution contains 10 mM citrate phosphate.

6. The pharmaceutical composition according to claim 1, wherein the sucrose is present in a concentration of 5% by weight / volume to 8% by weight / volume.

7. The pharmaceutical composition according to claim 1, wherein the sucrose is present at a concentration of 8% by weight / volume.

8. The pharmaceutical composition according to claim 1, wherein the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

9. The pharmaceutical composition according to claim 1, wherein the surfactant is polysorbate 80 and is present at a concentration of 0.02% by weight / volume.

10. The aforementioned pharmaceutical composition has the following characteristics: The pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at -20°C; The pharmaceutical composition is stable for at least 12 months, 18 months, or 24 months when stored at 2 to 8°C; The concentration of the fusion protein in the pharmaceutical composition remains substantially the same for at least 12 months, 18 months, or 24 months when stored at -20°C or 2 to 8°C; The concentration of the fusion protein in the pharmaceutical composition shall not decrease by more than 0.01%, more than 0.02%, more than 0.03%, more than 0.04%, more than 0.05%, more than 0.06%, more than 0.07%, more than 0.08%, more than 0.09%, more than 0.1%, more than 0.2%, more than 0.3%, more than 0.4%, more than 0.5%, more than 0.6%, more than 0.7%, more than 0.8%, more than 0.9%, or more than 1% after storage at -20°C or 2 to 8°C for 12 months, 18 months, or 24 months; The pharmaceutical composition is stable after one, two, three, four, or five freeze-thaw cycles; The fusion protein retains bifunctional activity, as measured by bifunctional enzyme-linked immunosorbent assay (ELISA), for at least 12 months, 18 months, or 24 months when stored at -20°C or 2–8°C; and / or The pharmaceutical composition comprises the fusion protein in aggregate form in an amount of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%; The pharmaceutical composition according to claim 1, comprising one or more of the following.

11. The aforementioned pharmaceutical composition After storage at -20°C or 2-8°C for 12 months, 18 months, or 24 months, compared to the control formulation, a. Increased shelf life, b. Increased temperature stability, c. reduced aggregate formation, d. Increased chemical stability, e. Reduced fragmentation, and / or f. Reduced viscosity The pharmaceutical composition according to claim 1, having at least one characteristic selected from the group consisting of the following.

12. The pharmaceutical composition according to claim 1, wherein the fusion protein is present at a concentration of 25 to 50 mg / ml.

13. The pharmaceutical composition according to claim 1, wherein the fusion protein is present at a concentration of 50 mg / ml.

14. The pharmaceutical composition according to Claim 1, a. The fusion protein at a concentration of 25-50 mg / ml, b. 5 mM to 20 mM citrate phosphate buffer, c. Sucrose in a concentration of 6% by weight / volume to 10% by weight / volume, d. Polysorbate 80 in a concentration of 0.01 wt / volume% to 0.05 wt / volume%, Includes, The pharmaceutical composition has a pH of 5.5 to 6.

5. Pharmaceutical composition.

15. A pharmaceutical composition according to claim 1, a. The fusion protein at 50 mg / mL, b. 10 mM citrate phosphate buffer, c. Sucrose at 8% by weight / volume, d. 0.02 wt / volume% polysorbate 20 and Includes, The pharmaceutical composition has a pH of 6.0 ± 0.

5. Pharmaceutical composition.

16. The pharmaceutical composition according to claim 2, a. The fusion protein at 50 mg / mL, b. 10 mM citrate phosphate buffer, c. Sucrose at 8% by weight / volume, d. 0.02 wt / volume% polysorbate 20 and Includes, The pharmaceutical composition has a pH of 6.0 ± 0.

5. Pharmaceutical composition.

17. The pharmaceutical composition according to claim 3, a. The fusion protein at 50 mg / mL, b. 10 mM citrate phosphate buffer, c. Sucrose at 8% by weight / volume, d. 0.02 wt / volume% polysorbate 20 and Includes, The pharmaceutical composition has a pH of 6.0 ± 0.

5. Pharmaceutical composition.

18. The pharmaceutical composition according to claim 1, for use in a method for treating human cancer in subjects with cancer.

19. The pharmaceutical composition according to claim 18, wherein the cancer is a solid tumor.

20. The pharmaceutical composition according to claim 18, wherein the cancer is selected from the group consisting of eye, stomach, colon, rectum, colorectal, breast cancer, anal cancer, pancreatic cancer, thyroid cancer, liver cancer, ovarian cancer, lung cancer, skin cancer, brain cancer, spinal cord cancer, head cancer, and neck cancer.

21. The pharmaceutical composition according to claim 18, wherein the cancer is head and neck cancer.

22. The pharmaceutical composition according to claim 18, wherein the cancer is head and neck squamous cell carcinoma (HNSCC).

23. The pharmaceutical composition according to claim 18, wherein the cancer is anal cancer.

24. The pharmaceutical composition according to claim 18, wherein the cancer is squamous cell carcinoma (SCCAC) of the anal canal.

25. A method for manufacturing a pharmaceutical composition, a. A step of culturing mammalian cells in which one or more nucleic acids encoding a fusion protein comprising a targeting portion and an immunomodulatory portion are stably incorporated into their genome, in a cell culture medium such that the cells secrete the fusion protein into the cell culture medium, wherein (i) the targeting portion specifically binds to hEGFR, and (ii) the immunomodulatory portion comprises the amino acid sequence of the extracellular domain of hTGFβRII, b. A step of purifying the fusion protein from the cell culture medium, c. The step of preparing the pharmaceutical composition described in claim 1. A method that includes this.

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