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 TO RELATED APPLICATIONS This application claims priority to Indian Provisional Application No. IN202011054539, filed December 15, 2020, the entire 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 connected or connected via linkers. The instability of antibodies and fusion proteins makes it difficult to develop stable formulations suitable for delivery to subjects. For example, these protein preparations may have a short shelf life, and the proteins may lose 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, precipitation, and adsorption. Despite their general structural similarities, developing formulations of different monoclonal antibodies has not been simple due to their unique and unpredictable behavior in liquid formulations. This unpredictability is even greater for multifunctional fusion proteins (e.g., fusion proteins comprising an antibody and another protein component) due to, for example, the relative significance of primary sequence, structure, linkage, multifunctionality, and degradation pathways (e.g., denaturation, aggregation, surface adsorption, deamidation, oxidation, isomerization, fragment formation, etc.) Thus, there remains a need for novel stable formulations of multifunctional fusion proteins (e.g., those described herein) that, for example, exhibit stability, low or undetectable levels of physical or chemical degradation, and little or no loss of multifunctional biological activity, even after extended storage. Summary of the Invention

[0003] The present invention addresses the above-mentioned needs by providing stable liquid pharmaceutical formulations of multifunctional fusion proteins (e.g., BCA101), as further described below. Specifically, the present invention provides stable liquid pharmaceutical formulations of the bifunctional fusion proteins disclosed herein. The formulations of the present invention are useful for administration (e.g., by intravenous administration) to mammals, particularly humans suffering from cancer.

[0004] Accordingly, in one aspect, 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-adjusting agent present at a concentration of about 4% w / v to about 10% w / v; wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 7.0.

[0005] Accordingly, in one aspect, 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 adjusting agent present at a concentration of about 4% w / v to about 10% w / v, wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 7.0.

[0006] In some embodiments, the buffer is a citrate phosphate buffer, a citrate buffer, a succinate buffer, or a histidine buffer. In some embodiments, the buffer is a citrate phosphate buffer. In some embodiments, the buffer is present at a concentration 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 a concentration of about 5 mM to about 15 mM. In some embodiments, the buffer is present at a concentration 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 tonicity adjusting agent is a sugar. In some embodiments, the tonicity adjusting agent is a disaccharide. In some embodiments, the tonicity adjusting agent is sucrose or trehalose. In some embodiments, the tonicity adjusting agent is sucrose. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5% to about 10% wt / vol, 6% to about 10% wt / vol, 7% to about 10% wt / vol, 8% to about 10% wt / vol, 5% to about 9% wt / vol, 5% to about 8% wt / vol, 6% to about 9% wt / vol, 6% to about 8% wt / vol, 7% to about 9% wt / vol, or 7% to about 8% wt / vol. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5% to about 8% wt / vol. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5%, 6%, 7%, 8%, 9%, or 10% w / v. In some embodiments, the tonicity adjusting agent is present at a concentration of about 8% w / v. In some embodiments, the tonicity adjusting agent is sucrose and is present at a concentration of about 8% w / v.

[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 0.005-0.1% w / v. In some embodiments, the surfactant is about 0.01 to 0.1% w / v, 0.02 to 0.1% w / v, 0.01 to 0.9% w / v, 0.01 to 0.8% w / v, 0.01 to 0.7% w / v, 0.01 to 0.6% w / v, 0.01 to 0.5% w / v, 0.01 to 0.4% w / v, 0.01 to 0.3% w / v, 0.01 to 0.2% w / v, 0.01 to 0.1% w / v, 0.02 to 0.9% w / v, 0.02 to 0.8% w / v, 0.02 to 0.7% w / v, 0.02 to 0.7% w / v, 0.02 to 0.8% w / v, 0.02 to 0.7% w / v, 0.02 ...8% w / v, 0.02 to 0.7% w / v, 0.02 to 0.8% w / v, 0.02 to 0.7% w / v, 0.02 to 0.8% w / v, 0.02 to 0 It is present in a concentration 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 a concentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% w / v. In some embodiments, the surfactant is present at a concentration of about 0.02% w / v. In some embodiments, the surfactant is polysorbate 20 and is present at a concentration of about 0.02% w / v.

[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 an osmolality of about 150 mOsmol / kg to about 400 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 an osmolality of about 250 mOsmol / kg to about 350 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has an osmolality of about 250 mOsmol / kg, 300 mOsmol / kg, or 300 mOsmol / kg. In some embodiments, the liquid pharmaceutical composition has an 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 to 1, 2, 3, 4, or 5 freeze and thaw cycles.

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

[0016] In some embodiments, the liquid pharmaceutical composition contains less than about 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% of the fusion protein in aggregate form.

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

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

[0019] In some embodiments, the fusion protein is present at a concentration of about 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 a concentration of about 20-30 mg / ml. In some embodiments, the fusion protein is present at a concentration of about 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 a concentration of about 25 mg / ml.

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

[0021] In some embodiments, the antibody, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a VH comprising VH CDR1, VH CDR2, and VH CDR3, wherein (a) VH CDR1 comprises an amino acid sequence 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 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 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, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein (a) the VL CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4; (b) the VL CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5; and (c) the VL CDR3 comprises an amino acid sequence 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, or functional fragment or functional variant thereof, that specifically binds to hEGFR 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, or functional fragment or functional variant thereof, that specifically binds to hEGFR 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, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a heavy chain comprising an amino acid sequence 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 functional fragment or functional variant thereof, that specifically binds to hEGFR consists of 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:10.

[0027] In some embodiments, the antibody, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a heavy chain consisting of an amino acid sequence 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 functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a heavy chain consisting of an amino acid sequence 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 functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a light chain comprising an amino acid sequence 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 functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a light chain consisting of an amino acid sequence 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 functional fragment or functional variant thereof, that specifically binds to hEGFR comprises cetuximab or panitumumab, or a functional fragment or functional variant of any of the foregoing.

[0032] In some embodiments, the immunomodulatory portion comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23. In some embodiments, the immunomodulatory portion consists of an amino acid sequence 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 moiety is indirectly fused to the targeting moiety, hi some embodiments, the immunomodulatory moiety is indirectly fused to the targeting moiety via a peptide linker.

[0034] In some embodiments, the immunomodulatory moiety is indirectly fused to the targeting moiety via a peptide linker of sufficient length to allow the immunomodulatory moiety and the targeting moiety to simultaneously bind to their respective targets. 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 moiety is fused to the C-terminus of the targeting moiety. In some embodiments, the immunomodulatory moiety is fused to the N-terminus of the targeting moiety.

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

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

[0038] In some embodiments, the targeting moiety is an antibody that specifically binds to hEGFR, comprising a heavy chain comprising 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 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11; the immunomodulatory moiety comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23; and the N-terminus of the immunomodulatory moiety is indirectly fused to the C-terminus of the heavy chain or the light chain via a linker, wherein the linker comprises 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 targeting moiety is an antibody that specifically binds to hEGFR, comprising a heavy chain comprising 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 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11; and the immunomodulatory moiety comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 23, and the N-terminus of the immunomodulatory moiety is indirectly fused to the C-terminus of the light chain via a linker, wherein the linker comprises 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 targeting moiety comprises an antibody comprising a heavy chain comprising 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 comprising an amino acid sequence 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 aspect, provided herein is 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 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% w / v to about 10% w / v sucrose, wherein the liquid pharmaceutical composition has a pH of about 5.5 to about 6.5.

[0043] In some embodiments, the targeting moiety comprises an antibody comprising a heavy chain comprising 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 comprising an amino acid sequence 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 about 25 mg / ml.

[0045] In some embodiments, the liquid pharmaceutical composition further comprises about 0.01-0.05% w / v polysorbate 20.

[0046] In one aspect, provided herein is a liquid pharmaceutical composition comprising: (a) a fusion protein comprising a targeting portion and an immunomodulatory portion, wherein (i) the targeting portion specifically binds to hEGFR and (ii) the immunomodulatory portion comprises an amino acid sequence of the extracellular domain of hTGFβRII; (b) about 10 mM citrate phosphate buffer; and (c) about 8% weight / volume 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% w / v polysorbate 20.

[0048] In some embodiments, the targeting moiety comprises an antibody comprising a heavy chain comprising 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 comprising an amino acid sequence 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 about 25 mg / ml.

[0050]

[0013] In one embodiment, provided herein is a liquid pharmaceutical composition comprising: (a) about 25 mg / mL of a fusion protein comprising a targeting moiety and an immunomodulatory moiety, wherein the targeting moiety comprises an antibody comprising a heavy chain comprising 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 comprising 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% w / v sucrose; and (d) about 0.02% w / v polysorbate 20, wherein the liquid pharmaceutical composition has a pH of about 6.0.

[0051] In one aspect, provided herein is a method of treating human cancer in a subject having cancer, the method comprising 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 at a dose of about 10 mg to 2000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 20 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 30 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 40 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 50 mg to 1000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 100 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 900 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 800 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 700 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 800 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 700 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 600 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 500 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 400 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 300 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 100 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 10 mg to 50 mg.

[0054] In some embodiments, the fusion protein is administered to the human subject at a dose of about 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, or 2000 mg. In some embodiments, the fusion protein is administered to the human subject at a dose of about 64 mg, 240 mg, 800 mg, or 1600 mg.

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

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

[0057] In some embodiments, the administering step comprises intravenously injecting 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 contain genomic amplification of the EGFR gene, as detected, for example, by biopsy or fluorescence in situ hybridization.

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

[0061] In some embodiments, 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 cancer, head cancer, and neck cancer.

[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 detectable levels of programmed death-ligand 1, as determined by biopsy. In some embodiments, the SqCLC comprises cancer cells that contain 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 cancer. In some embodiments, the spinal 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 anaplastic thyroid cancer (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 aspect, provided herein is a method of making a liquid pharmaceutical composition, the method comprising: (a) culturing mammalian cells, having one or more nucleic acids stably integrated into their genomes, encoding a fusion protein comprising a targeting moiety and an immunomodulatory moiety, 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 a pharmaceutical composition described herein. [Brief explanation of the drawings]

[0065] [Figure 1] FIG. 1 is a schematic diagram showing the development strategy for the formulations described herein. [Figure 2] FIG. 1 is a schematic diagram outlining the pH screening study described in Example 1 and test BCA101 formulations at pH 5.0, 5.5, 6.0, and 6.5. [Figure 3] 1 is a dot graph showing the percentage of high molecular weight proteins (HMWPs) in the bulk tangential flow filtered composition (TFF) and the final drug product (FDP) at each test pH (5.0, 5.5, 6.0, and 6.5). [Figure 4] 1 is a dot graph showing the percentage of low monomeric protein in the bulk TFF composition and FDP at each tested pH (5.0, 5.5, 6.0, and 6.5). [Figure 5] 1 is a dot graph showing the percentage of low molecular weight proteins (LMWP) in the bulk TFF composition and FDP at each test pH (5.0, 5.5, 6.0, and 6.5). [Figure 6-1] 1 is a series of line graphs showing the results of differential scanning calorimetry (DSC) analysis, as described in Example 1, for each BCA101 test formulation evaluated. [Figure 6-2] (Same as above.) [Figure 7] 7 is a dot graph summarizing the results of the DSC analysis described in Example 1 for each test formulation shown in FIG. 6. [Figure 8] Figure 8A is a line graph showing percent HMWP at 40° C. for test formulations at pH 6.0 and 6.5. Figure 8B is a dot graph showing percent HMWP slope / week at 40° C. for test formulations at pH 6.0 and 6.5. [Figure 9] Figure 9A is a line graph showing percent monomer at 40° C. for test formulations at pH 6.0 and 6.5. Figure 9B is a dot graph showing percent monomer slope / week at 40° C. for test formulations at pH 6.0 and 6.5. [Figure 10] Figure 10A is a line graph showing percent LMWP at 40° C. for test formulations at pH 6.0 and 6.5. Figure 10B is a dot graph showing percent LMWP slope / week at 40° C. for test formulations at pH 6.0 and 6.5. [Figure 11] FIG. 1 is a schematic diagram showing the process of the buffer screening study described in Example 1 and the composition of the test formulations evaluated. [Figure 12] Figure 12A is a line graph showing the results of DSC analysis for a citrate buffer formulation having a pH of 6.0, and Figure 12B is a line graph showing the results of DSC analysis for a citrate buffer formulation having a pH of 6.5. [Figure 13] Figure 13A is a line graph showing the results of DSC analysis for a succinate buffer formulation having a pH of 6.0, and Figure 13B is a line graph showing the results of DSC analysis for a succinate buffer formulation having a pH of 6.5. [Figure 14] Figure 14A is a line graph showing the results of DSC analysis for a histidine buffer formulation having a pH of 6.0, and Figure 14B is a line graph showing the results of DSC analysis for a histidine buffer formulation having a pH of 6.5. [Figure 15]Figure 15A is a line graph showing the results of DSC analysis for a citrate phosphate buffer formulation having a pH of 6.0, and Figure 15B is a line graph showing the results of DSC analysis for a citrate phosphate buffer formulation having a pH of 6.5. [Figure 16] 12A-15B are dot graphs summarizing the DSC analysis data presented in FIGS. [Figure 17] FIG. 1 is a schematic diagram showing the tonicity modifying agent screening process described in Example 1, testing test formulations containing sucrose or trehalose. [Figure 18] 1 is a photocopy of a BCA101 fusion protein in a formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0), comparing the color of the BCA101 liquid formulation with a Pharmacopoeia (Ph.Eu.2.2.2) color standard solution. [Figure 19] 1 is a table showing the absorbance at 506 nm of BCA101 fusion protein in a formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) for toxicology study batches, internal reference standard batches (IRS batches), and dose range discovery batches. [Figure 20] 1 is a photocopy of a BCA101 fusion protein in a formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0), comparing the clarity and turbidity of the BCA101 liquid formulation with a pharmacopoeia standard (formazin suspension, Ph.Eu.2.2.1). [Figure 21]1 is a table showing the turbidity in nephelometric turbidity units (NTU) of a BCA101 fusion protein in a formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0). Batches analyzed before filtration are designated "BF," and batches analyzed after filtration are designated "AF." The assay was performed in accordance with Ph.Eur.2.2.1, the entire contents of which are incorporated herein by reference. [Figure 22] 1 is a series of dot graphs showing the pH, osmolality, protein concentration, and bifunctional capacity of BCA101 drug substance (DS) formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over the course of 24 months stored in 5 mL Celsius bags at −20° C. Samples were analyzed at the initial time point, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 23] 1 is a series of dot graphs showing the pH, osmolality, protein concentration, and bifunctional capacity of BCA101 drug product (DP) formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over the course of 24 months stored in 5 mL Celsius bags at −20° C. Samples were analyzed at the initial time point, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 24] 24 is a table showing a comparison of long-term stability data for BCA101 DS presented in FIG. 22 and DP presented in FIG. 23. [Figure 25]1 is a series of dot graphs showing the percent LMWP, percent monomer, and percent HMWP of BCA101 DS formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over the course of 24 months stored in 5 mL Celsius bags at −20° C. Samples were analyzed at the initial time point, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 26] 1 is a series of dot graphs showing percent LMWP, percent monomer, and percent HMWP of BCA101 DP formulated in a liquid formulation containing 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0) over the course of 24 months stored in 5 mL Celsius bags at −20° C. Samples were analyzed at the initial time point, 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, and 24 months. [Figure 27] 27 is a table showing a comparison of long-term stability data for BCA101 DS presented in FIG. 25 and DP presented in FIG. 26. [Figure 28] FIG. 1 shows an exemplary manufacturing process of the present disclosure for producing a BCA101 formulation described herein, e.g., a formulation comprising 25 mg / ml BCA101, 8.0% w / v sucrose, 0.02% w / v polysorbate 20, and 10 mM citrate phosphate buffer (pH 6.0). [Figure 29] 1 is a line graph showing the pH trend of BCA100 drug substance (DS) stability data over 24 months of storage at -20±5°C. [Figure 30] 1 is a line graph showing the osmolality trend of BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. [Figure 31]1 is a line graph showing the protein concentration trend of BCA100 drug substance (DS) stability data over 24 months of storage at -20±5°C. [Figure 32] 1 is a line graph showing the trend of percent high molecular weight protein for BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. [Figure 33] 1 is a line graph showing the percent monomeric protein trend of BCA100 drug substance (DS) stability data upon storage at −20±5° C. over 24 months. [Figure 34] 1 is a line graph showing the trend of percent low molecular weight protein for BCA100 drug substance (DS) stability data over 24 months of storage at -20±5°C. [Figure 35] 1 is a line graph showing the trend of percent total protein in the pre-peak of BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. [Figure 36] 1 is a line graph showing the trend of percent total protein post-peak for BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. [Figure 37] 1 is a line graph showing the trend of percent total protein in the main peak of BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. [Figure 38] 1 is a line graph showing the trend in relative potency of BCA100 drug substance (DS) stability data over 24 months of storage at −20±5° C. as measured by bifunctional ELISA. [Figure 39] 1 is a line graph showing the trend in relative potency of BCA100 drug substance (DS) stability data over 24 months of storage at -20±5° C. as measured by the inhibition of proliferation (IOP) assay. [Figure 40] 1 is a line graph showing pH trend of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 41]1 is a line graph showing the osmolality trend of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 42] 1 is a line graph showing protein concentration trends of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 43] 1 is a line graph showing the trend of collected volume of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 44] 1 is a line graph showing the trend of percent high molecular weight protein for BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 45] 1 is a line graph showing the percent monomeric protein trend of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 46] 1 is a line graph showing the trend of percent low molecular weight protein for BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 47] 1 is a line graph showing the trend of percent total protein in the pre-peak of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 48] 1 is a line graph showing the trend of percent total protein post-peak for BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 49] 1 is a line graph showing the trend of percent total protein in the main peak of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. [Figure 50] 1 is a line graph showing the trend of relative potency of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. as measured by bifunctional ELISA. [Figure 51]1 is a line graph showing the trend of relative potency of BCA100 drug product (DP) stability data over 24 months of storage at 5±3° C. as measured by the inhibition of proliferation (IOP) assay. [Figure 52] iCE electropherograms of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (top panel) and BCA101 DS GF19000040 (bottom panel). The left and right pI markers correspond to 4.05 and 8.4, respectively. [Figure 53] Figure 1 shows intact mass spectra showing the intact mass of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (top panel) and BCA101 DS GF19000040 (bottom panel) using MALDI-TOF. [Figure 54] UV chromatograms of peptides generated from tryptic digestion of BCA101 DS batch BL.14.0901 / R / 17 / 021 / F DS (top panel) and BCA101 DS batch GF19000040 (bottom panel) are shown. [Figure 55] 1 shows the MS2 tandem spectrum of the N-terminus of the heavy chain of BCA101 DS batch GF19000040. [Figure 56] 1 shows the MS2 tandem spectrum of the C-terminus of the heavy chain of BCA101 DS batch GF19000040. [Figure 57] 1 shows the MS2 tandem spectrum of the N-terminus of the light chain of BCA101 DS batch GF19000040. [Figure 58] 1 shows the MS2 tandem spectrum of the C-terminus of the linker of BCA101 DS batch GF19000040. [Figure 59] 1 shows the MS spectrum of the C-terminus of the TGFβRII chain of BCA101 DS batch GF19000040. [Figure 60] An overlay of the far-UV CD spectrum of BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS is shown. [Figure 61]An overlay of the near-UV CD spectrum of BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS is shown. [Figure 62] The UV chromatogram profile of tryptic non-reduced peptides of BCA101 DS batch GF19000040 is shown together with BL.14.0901 / R / 17 / 021 / F DS. [Figure 63] An overlay of the NP-HPLC profile of N-glycans from BCA101 DS batch GF19000040 with BL.14.0901 / R / 17 / 021 / F DS is shown. The N-glycans printed here were identified by MS. Identification of the "other species" is ongoing. [Figure 64] RP-HPLC chromatogram of sialic acid estimates of BCA101 DS batch GF19000040 overlayed with NGNA, NANA standards, and the corresponding buffer blank. DETAILED DESCRIPTION OF THE INVENTION

[0066] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0067] overview The present disclosure provides, inter alia, pharmaceutical formulations of the bifunctional fusion proteins described herein that allow for 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., the targeting moiety) has an acidic pI, while the second functional aspect (e.g., the immunomodulatory domain) has a basic pI. In some embodiments, an 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 driven by hEGFR.

[0068] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. In this application, the use of the singular includes the plural unless otherwise indicated.

[0069] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0070] Where embodiments are described herein using the word "comprising," it is understood that similar embodiments separately described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0071] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0072] The term "and / or," as used herein, is to be construed as a specific disclosure that each of the two specified features or components may or may not be accompanied by one another. Thus, 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 encompass 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 commonly understood by those skilled in the art to which this disclosure pertains.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 those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0074] Units, prefixes, and symbols are denoted in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0075] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include every integer and, where appropriate, fractional values ​​thereof (such as tenths and hundredths of integers) within the recited range, unless otherwise indicated.

[0076] The term "about" or "essentially consisting of" means a value or composition that is within an acceptable range of error for a particular value or composition, as determined by one of ordinary skill in the art, which will depend, in part, on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially consisting of" can mean within one or more standard deviations, as is customary in the art. Alternatively, "about" or "essentially consisting of" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise indicated, the meaning of "about" or "essentially consisting of" should be assumed to be within an acceptable range 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 such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human.

[0078] As used herein, the term "administering" refers to the physical introduction of a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or altered in the subject's body to produce the therapeutic agent in vivo) into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Therapeutic agents may be administered via parenteral routes or orally. Other parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, intravaginal, rectal, sublingual, or topical. Also, administration can be performed, for example, once, multiple times, and / or over one or more extended periods of time.

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

[0080] A "therapeutically effective amount" or "therapeutically effective dose" of a therapeutic agent is any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression as indicated by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0081] The term "antibody" is used herein in the broadest sense and includes fully assembled antibodies, functional antibody fragments and functional variants thereof capable of binding to antigen (e.g., Fab, F(ab')2, Fv, single-chain 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 antigen (e.g., recombinant fibronectin domains), and recombinant polypeptides comprising the foregoing. Unless otherwise indicated, references to the numbering of specific amino acid residue positions in an antibody are in accordance with the EU numbering system as set forth in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) ("Kabat"), the entire contents of which are incorporated herein by reference.

[0082] As used herein, the term "variable region" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a native antibody 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 of the regions of an antibody variable domain whose sequence is hypervariable and forms a structurally defined loop ("hypervariable loop"). Generally, a native four-chain 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 these definitions include overlapping or subsets of amino acid residues when compared with each other. Regardless, the application of either definition to refer to an antibody CDR is intended to be within the scope of the term as defined and used herein. Those skilled in the art can routinely determine which residues constitute a particular CDR based on the amino acid sequence of the antibody variable region. Unless otherwise specified, CDRs are defined according to the Kabat system.

[0084] The term "fusion protein" and its grammatical equivalents used herein refer to a protein comprising an amino acid sequence derived from at least two separate proteins.The amino acid sequences of at least two separate proteins may be directly connected through a peptide bond, or may be operably connected through an amino acid linker.Therefore, the term fusion protein encompasses, for example, an embodiment in which the amino acid sequence of protein A is directly connected to the amino acid sequence of protein B through a peptide bond (protein A-protein B), and, for example, an embodiment in which the amino acid sequence of protein A is operably connected to the amino acid sequence of protein B through an amino acid linker (protein A-linker-protein B).

[0085] As used herein, the term "fused" and its grammatical equivalents refer to the operative connection of an amino acid sequence derived from one protein to an amino acid sequence derived from another protein. The term fused encompasses both a direct connection of two amino acid sequences via a peptide bond and an indirect connection via an amino acid linker.

[0086] As used herein, the term "modification" in relation to a nucleic acid sequence refers to a nucleic acid sequence that contains at least one substitution, addition, or deletion of a nucleotide compared to a reference nucleic acid sequence. As used herein, the term "modification" in relation to an amino acid sequence refers to an amino acid sequence that contains at least one substitution, addition, or deletion of an amino acid residue compared to a reference amino acid sequence. Naturally occurring amino acid derivatives are not considered modified amino acids for purposes of determining the percent identity of two amino acid sequences. For example, a naturally occurring modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue is not considered an amino acid modification for purposes of determining the percent identity of two amino acid sequences. Furthermore, for example, a naturally occurring modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue would not be considered an amino acid "modification" as defined herein. Modifications can include the inclusion of non-naturally occurring amino acid residues.

[0087] The term "identical" or "percent identity" in relation to a nucleic acid sequence or amino acid sequence refers to at least two nucleic acid or at least two amino acid sequences or subsequences, respectively, being compared and aligned for maximum correspondence, having a specified percentage of identical nucleotides or amino acids, as determined by using a sequence comparison algorithm or by visual inspection.For sequence comparison, typically, one sequence serves as a reference sequence, to which a test sequence is compared.When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated if necessary, and the program parameters of the sequence algorithm are designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters. Examples of suitable algorithms for determining percent 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 percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, typically, only perfect matches are counted. As described above, the percent identity is based on the amino acid match in the smaller of the two proteins.

[0088] A "stable" pharmaceutical composition is one in which the protein therein essentially retains physical and / or chemical stability and / or biological activity during processing (e.g., ultrafiltration, diafiltration, other filtration steps, vial filling), shipping, and / or storage of the drug substance and / or drug product comprising the fusion protein described herein. Furthermore, the physical, chemical, and biological stability of the protein in the formulation embodies the "stability" of a protein formulation, e.g., a 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 it shows minimal changes in secondary and / or tertiary structure (i.e., essential structure), or signs of aggregation and / or precipitation and / or denaturation, as determined by visual inspection of color and / or clarity, or by UV light scattering, size-exclusion high-performance liquid chromatography, or other suitable methods. Protein physical instability, i.e., loss of physical stability, can be caused by oligomerization, which leads to the formation of dimers and higher-order aggregates, subvisible and visible particles, and precipitation. The degree of physical degradation can be confirmed 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 subvisible particles can be quantified using light scattering, light obscuration, or other suitable techniques.

[0090] A protein retains its "chemical stability" in a pharmaceutical composition if its chemical stability at a given time is such that no covalent bonds are created or broken, and no changes occur to the primary structure of the protein components, such as the fusion proteins described herein. Changes in primary structure can result in alterations to the secondary, tertiary, and / or quaternary structure of the protein, leading to the formation of aggregates or the reversal of already formed aggregates. Typical chemical modifications include isomerization, deamination, N-terminal cyclization, backbone hydrolysis, methionine oxidation, tryptophan oxidation, histidine oxidation, beta-elimination, disulfide formation, disulfide scrambling, disulfide cleavage, and other changes that result in changes to primary structure, including D-amino acid formation. Chemical instability, i.e., loss of chemical stability, can be examined by various techniques, including ion-exchange chromatography, capillary isoelectric focusing, peptide digest analysis, and several types of mass spectrometry. Chemical stability can be assessed by detecting and quantifying chemically modified forms of the protein. Chemical modifications can include size modifications (e.g., clipping), which can be assessed 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 modifications (e.g., resulting from deamidation), which can be assessed 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 a change in biological activity, either an increase or a decrease in the desired biological activity, depending on the modification and the protein being modified. A protein retains its "biological activity" in a pharmaceutical composition if the biological activity of the protein at a given time is at least 30% of the biological activity exhibited at the time the pharmaceutical formulation was prepared. Activity is considered to be decreased if the activity falls below 70% of its starting value. Biological assays can include both in vivo and in vitro-based assays, such as 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 the bifunctional ELISA, a plate coated with recombinant hEGFR Fc was 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, which will only give a signal if both arms are intact.

[0092] Pharmaceutical Composition In certain aspects, described herein are pharmaceutical compositions (e.g., liquid pharmaceutical compositions) comprising a fusion protein (e.g., a fusion protein described herein), a buffer, and a tonicity adjusting agent, and having a preselected pH. In some embodiments, the pharmaceutical composition comprises one or more additional 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 a citrate phosphate buffer, a citrate buffer, a succinate buffer, or a histidine buffer. In some embodiments, the buffer is a citrate phosphate buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is a succinate buffer. In some embodiments, the buffer is a histidine buffer.

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

[0095] In certain embodiments, the buffer is a citrate phosphate buffer present at a concentration of about 5 mM to about 30 mM, 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 a citrate phosphate buffer present at a concentration of about 5 mM to about 15 mM. In some embodiments, the buffer is a citrate phosphate buffer present at a concentration of about 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 about 10 mM. In some embodiments, the pharmaceutical composition has a pH of about 6.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of about 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] Tonicity adjuster In some embodiments, the pharmaceutical composition includes a tonicity adjusting agent such that the formulation has a final osmolality of about 200-400 mOsmol / kg. In some embodiments, the pharmaceutical composition includes a tonicity adjusting agent such that the formulation has a final osmolality of about 250-350 mOsmol / kg. In some embodiments, the pharmaceutical composition includes a tonicity adjusting agent such that the formulation has a final osmolality of about 300 mOsmol / kg.

[0098] In some embodiments, the tonicity adjusting agent comprises sucrose, trehalose, sorbitol, mannitol, or glycerol. In some embodiments, the tonicity adjusting agent is sucrose. In some embodiments, the tonicity adjusting agent is trehalose. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5% to about 10% w / v, 6% to about 10% w / v, 7% to about 10% w / v, 8% to about 10% w / v, 5% to about 9% w / v, 5% to about 8% w / v, 6% to about 9% w / v, 6% to about 8% w / v, 7% to about 9% w / v, or 7% to about 8% w / v. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5% to about 8% w / v. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5%, 6%, 7%, 8%, 9%, or 10% w / v, hi some embodiments, the tonicity adjusting agent is present at a concentration of about 8% w / v.

[0099] In some embodiments, the tonicity adjusting agent is sucrose at a concentration such that the formulation has a final osmolality of about 300 mOsmol / kg. In some embodiments, the tonicity adjusting agent comprises sucrose at a concentration of about 5% to about 10%, 6% to about 10%, 7% to about 10%, 8% to about 10%, 5% to about 9%, 5% to about 8%, 6% to about 9%, 6% to about 8%, 7% to about 9%, or 7% to about 8% wt / vol. In some embodiments, the tonicity adjusting agent comprises sucrose at a concentration of about 5% to about 8% wt / vol. In some embodiments, the tonicity adjusting agent is present at a concentration of about 5%, 6%, 7%, 8%, 9%, or 10% w / v, hi some embodiments, the tonicity adjusting agent is sucrose at a concentration of about 8% w / v.

[0100] surfactants In some embodiments, the pharmaceutical composition includes a surfactant. In some embodiments, the surfactant is a non-ionic 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)), such as 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 concentration of the surfactant is about 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%, %, 0.02 to 0.6 wt / vol%, 0.02 to 0.5 wt / vol%, 0.02 to 0.4 wt / vol%, 0.02 to 0.3 wt / vol%, 0.02 to 0.2 wt / vol%, 0.02 to 0.1 wt / vol%, 0.005 to 0.9 wt / vol%, 0.005 to 0.8 wt / vol%, 0.005 to 0.7 wt / vol%, 0.005 to 0.6 wt / vol%, 0.005 to 0.5 wt / vol%, 0.005 to 0.4 wt / vol%, 0.005 to 0.3 wt / vol%, 0.005 to 0.2 wt / vol%, or 0.005 to 0.1 wt / vol%. In some embodiments, the surfactant concentration is about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% w / v. In some embodiments, the surfactant concentration is about 0.02% w / v.

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

[0103] Osmolality In some embodiments, the pharmaceutical composition has an 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 an osmolality of about 250 mOsmol / kg to about 350 mOsmol / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 250 mOsmol / kg, 300 mOsmol / kg, or 350 mOsmol / kg. In some embodiments, the pharmaceutical composition has an osmolality of about 300 mOsmol / kg.

[0104] Exemplary Functional Properties Stability and Storage The pharmaceutical composition can be stored in any suitable container known to those of skill in the art, such as a bag (e.g., a Celsius® bag or a Flexboy® bag) or a glass vial (e.g., a USP 10R glass vial). Containers suitable for storage at various temperatures (e.g., −80° C., −20° C., 2 to 8° C.) are known to those of skill in the art and can be selected appropriately. 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 to 8° C., the pharmaceutical composition is stored in a glass vial.

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

[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, wherein the freeze-thaw cycles include a 48-hour freezing cycle at -80°C or -20°C and a 4-hour thawing cycle 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, wherein the freeze-thaw cycles include a 48-hour freezing cycle at -80°C or -20°C and a 4-hour thawing cycle 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, wherein the freeze-thaw cycles include a 48-hour freezing cycle at -80°C or -20°C and a 4-hour thawing cycle at 25°C in an incubator. In some embodiments, the pharmaceutical composition is chemically and physically stable through at least 1, 2, 3, 4, or 5 freeze-thaw cycles, wherein the freeze-thaw cycles include a 48 hour freeze cycle at -80°C or -20°C and 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 less than about 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains less than about 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains less than about 5% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition comprises less than about 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregate form after storage for at least 12 months, 24 months, or 36 months at −20° C. In some embodiments, the pharmaceutical composition comprises less than about 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the fusion protein in aggregate form after storage for at least 12 months, 24 months, or 36 months at 2-8° C.

[0115] In some embodiments, the pharmaceutical composition contains 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% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains 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%, or no more than 10% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains no more than 5% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains 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% of the fusion protein in aggregate form after storage at -80°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains 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%, or no more than 10% of the fusion protein in aggregate form after storage at -80° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5% of the fusion protein in aggregate form after storage at -80° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains no more than 5% of the fusion protein in aggregate form after storage at -80° C. for at least 12 months, 24 months, or 36 months.In some embodiments, the pharmaceutical composition contains 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% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains 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%, no more than 20%, or no more than 30% of the fusion protein in aggregate form after storage at −20° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains 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%, or no more than 10% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains 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%, no more than 20%, or no more than 30% of the fusion protein in aggregate form after storage at −20° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains 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%, no more than 20%, or no more than 30% of the fusion protein in aggregate form. In some embodiments, the pharmaceutical composition contains no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5% of the fusion protein in aggregate form after storage at -20°C for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains no more than 5% of the fusion protein in aggregate form.In some embodiments, the pharmaceutical composition contains 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%, no more than 20%, or no more than 30% of the fusion protein in aggregate form after storage for at least 12 months, 24 months, or 36 months at −20° C. In some embodiments, the pharmaceutical composition contains 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% of the fusion protein in aggregate form after storage for at least 12 months, 24 months, or 36 months at 2-8° C. In some embodiments, the pharmaceutical composition contains 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%, or no more than 10% of the fusion protein in aggregate form after storage at 2-8° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains no more than 1%, no more than 2%, no more than 3%, no more than 4%, or no more than 5% of the fusion protein in aggregate form after storage at 2-8° C. for at least 12 months, 24 months, or 36 months. In some embodiments, the pharmaceutical composition contains no more than 5% of the fusion protein in aggregate form 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 the present disclosure. Aggregation can be assessed, for example, by size exclusion chromatography (SEC).

[0116] Stability can be measured by any assay known to those of skill in the art, including those described in Example 1 of the present 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] Bifunctionality of fusion proteins The fusion proteins described herein have two distinct functions: 1) specific binding to hEGFR and 2) specific 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%, 4%, 3%, 2%, 1%, or 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%, 4%, 3%, 2%, 1%, or 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%, 4%, 3%, 2%, 1%, or 0.5% of their hEGFR binding activity (e.g., as measured by ELISA) and lose less than 4%, 3%, 2%, 1%, or 0.5% of their hTGFβ binding activity (e.g., as measured by ELISA).

[0120] The bifunctionality of the fusion proteins described herein can be assessed by methods known in the art, including those described in Example 1 of the present disclosure. For example, the bifunctionality of the fusion proteins described herein can be assessed by two separate or combined ELISAs that assay for both functionalities of the fusion proteins described herein (i.e., 1) specifically binding to hEGFR, and 2) specifically binding to hTGFβ).

[0121] fusion proteins In certain embodiments, provided herein is a pharmaceutical composition comprising a multifunctional 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 with an acidic pI, wherein the membrane-bound target protein and the soluble target protein are different.

[0122] In certain embodiments, provided herein are pharmaceutical compositions comprising a multifunctional (bifunctional) 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.

[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 a 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 or panitumumab. In some embodiments, the anti-hEGFR antibody is a functional variant of cetuximab or 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 CDRs as cetuximab.

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

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

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

[0129] In some embodiments, the anti-hEGFR antibody comprises a variable light chain (VL) comprising three complementarity determining regions: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the anti-hEGFR antibody comprises a VL comprising a VL CDR1 comprising 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, a VL CDR2 comprising 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 a VL CDR3 comprising 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 a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1 with 0, 1, 2, or 3 amino acid modifications, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2 with 0, 1, 2, or 3 amino acid modifications, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3 with 0, 1, 2, or 3 amino acid modifications; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 4 with 0, 1, 2, or 3 amino acid modifications, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5 with 0, 1, 2, or 3 amino acid modifications, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6 with 0, 1, 2, or 3 amino acid modifications.

[0131] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 with one, two, or three amino acid modifications, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 with one, two, or three amino acid modifications, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 with one, two, or three amino acid modifications; and a VL comprising a VL CDR1 comprising 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, a VL CDR2 comprising 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 a VL CDR3 comprising 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.

[0132] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising 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 a VH CDR3 comprising an amino acid sequence 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 a VL comprising a VL CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising an amino acid sequence 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 a VH comprising a VH CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising 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 a VH CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 3; and the anti-hEGFR antibody comprises a VL CDR1 comprising 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 a VL CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5. and a VL comprising a CDR2, and a VL CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6.

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

[0136] In some embodiments, the anti-hEGFR antibody comprises a heavy chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 CDRs as panitumumab.

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

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

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

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

[0143] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 amino acid modifications, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13 with 0, 1, 2, or 3 amino acid modifications, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14 with 0, 1, 2, or 3 amino acid modifications; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 amino acid modifications, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, or 3 amino acid modifications, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, or 3 amino acid modifications.

[0144] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 12 with one, two, or three amino acid modifications, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 13 with one, two, or three amino acid modifications, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 14 with one, two, or three amino acid modifications; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15 with one, two, or three amino acid modifications, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 16 with one, two, or three amino acid modifications, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the amino acid sequence of SEQ ID NO: 17 with one, two, or three amino acid modifications.

[0145] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising 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 a VH CDR3 comprising an amino acid sequence 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 a VL comprising a VL CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, a VL CDR2 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and a VL CDR3 comprising an amino acid sequence 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 a VH comprising a VH CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising 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 a VH CDR3 comprising 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 a VL CDR1 comprising 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 a VL CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. and a VL comprising a CDR2, and a VL CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:17.

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

[0149] In some embodiments, the anti-hEGFR antibody comprises a heavy chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises an antibody in Table 1. In some embodiments, the anti-hEGFR antibody is an antibody in Table 1.

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

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

[0154] In some embodiments, the anti-hEGFR antibody comprises a VH comprising a VH CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH CDR1 in Table 1, a VH CDR2 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH CDR2 in Table 1, and a VH CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VH CDR3 in Table 1, a VL CDR1 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL CDR1 in Table 1, and a VL CDR2 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL CDR1 in Table 1. and a VL comprising a VL CDR2 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of CDR2, and a VL CDR3 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a VL CDR3 in Table 1.

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

[0157] [Table 1-1]

[0158] [Table 1-2]

[0159] [Table 1-3]

[0160] hTGFβ trap In certain embodiments, 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, the hTGFβRII ECD binds to at least one hTGFβ isoform. In some embodiments, the hTGFβRII ECD binds to hTGFβ1. In some embodiments, the hTGFβRII ECD binds to hTGFβ3. In some embodiments, the hTGFβRII ECD does not bind to hTGFβ2.

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

[0163] In some embodiments, the extracellular domain of hTGFβRII comprises a truncated portion of SEQ ID NO:23, which allows it to bind to hTGFβ. The extracellular domain of hTGFβRII may be truncated at the N-terminus, C-terminus, or both the N-terminus and C-terminus. The truncation may include deletion of 1 to 10 amino acids. The truncation may include deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The truncation may include 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 comprises an amino acid sequence 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 essentially of an amino acid sequence 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 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 moiety is operably linked to the C-terminus of the targeting moiety. In some embodiments, the immunomodulatory moiety is operably linked to the N-terminus of the targeting moiety.

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

[0168] Linker In some embodiments, the targeting portion and the immunomodulatory portion of the fusion protein are directly operably connected. In some embodiments, the targeting portion and the immunomodulatory portion of the fusion protein are indirectly operably connected. In some embodiments, the targeting portion and the immunomodulatory portion 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 that allows the immunomodulatory moiety and targeting moiety to be linked to their respective antigens can be used. Exemplary peptide linkers containing glycine and serine amino acids are provided in Table 3.

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

[0171] In some embodiments, the linker comprises an amino acid sequence 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 comprises an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 24, or the amino acid sequence of SEQ ID NO: 24 with one, two, or three amino acid modifications. In some embodiments, the linker consists essentially of an amino acid sequence 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 with one, two, or three amino acid modifications.

[0172] In some embodiments, the linker 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: 25. In some embodiments, the linker comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 25, or the amino acid sequence of SEQ ID NO: 25 with one, two, or three amino acid modifications. In some embodiments, the linker consists essentially 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 with one, two, or three amino acid modifications.

[0173] In some embodiments, the linker comprises an amino acid sequence 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 comprises an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 26, or the amino acid sequence of SEQ ID NO: 26 with one, two, or three amino acid modifications. In some embodiments, the linker consists essentially of an amino acid sequence 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 with one, two, or three amino acid modifications.

[0174] In some embodiments, the linker comprises an amino acid sequence 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 comprises an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 27. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 27, or the amino acid sequence of SEQ ID NO: 27 with one, two, or three amino acid modifications. In some embodiments, the linker consists essentially of an amino acid sequence 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 with one, two, or three amino acid modifications.

[0175] In some embodiments, the linker comprises an amino acid sequence 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 comprises an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence of SEQ ID NO: 28 with one, two, or three amino acid modifications. In some embodiments, the linker consists essentially of an amino acid sequence 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 with one, two, or three amino acid modifications.

[0176] [Table 3]

[0177] Exemplary Fusion Proteins Exemplary fusion proteins of the present disclosure are provided in Table 4.

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

[0179] In some embodiments, the fusion protein comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain comprising an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion protein comprises a light chain comprising an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the fusion protein comprises a heavy chain comprising an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain comprising an amino acid sequence 100% identical to the amino acid sequence of SEQ ID NO: 29.

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

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

[0183] In some embodiments, the fusion protein comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises 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 comprises 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 comprises 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 comprising 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 with 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 with 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 with one, two, or three amino acid modifications, and a light chain consisting of the amino acid sequence of SEQ ID NO: 29 with one, two, or three amino acid modifications.

[0186] In some embodiments, the fusion protein comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a heavy chain comprising an amino acid sequence 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 comprises a light chain comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprising an amino acid sequence 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 comprises a heavy chain consisting of an amino acid sequence 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 comprises a light chain consisting of an amino acid sequence 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 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 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 comprises a fusion protein described herein at a concentration of about 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 pharmaceutical composition comprises a fusion protein described herein at a concentration of about 20-30 mg / ml. In some embodiments, the pharmaceutical composition comprises a fusion protein described herein at a concentration of about 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 comprises a fusion protein described herein at a concentration of about 25 mg / ml.

[0206] In some embodiments, the fusion protein is BCA101 at a concentration of about 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 about 20-30 mg / ml. In some embodiments, the fusion protein is BCA101 and is present at a concentration of about 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 and is present at a concentration of about 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 comprising 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 comprising 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 comprising 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 comprising 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 aspects, the present invention provides a method for producing the pharmaceutical composition described herein.In some embodiments, the method comprises: culturing mammalian cells that contain one or more nucleic acids encoding the fusion protein described herein in cell culture medium, so that the cells secrete the fusion protein into cell culture medium; purifying the fusion protein from cell culture medium; and preparing the pharmaceutical composition described herein.

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

[0210] Any suitable mammalian cell can be used for expression of the fusion protein. For example, well-known mammalian cells include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic 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), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma 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 (MMT 060562, ATCC CCL51), TRI cells (Mather et al, 1982, Annals NY Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, and human hepatoma line (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.), minimal essential medium (MEM), (Sigma-Aldrich Co.), RPML 1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium (DMEM), Sigma-Aldrich Co., are suitable for culturing host cells. In addition, media such as those described in Ham et al., 1979, Meth. Enz. 58: 44; Barnes et al., 1980, Anal. Biochem. 102: 255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, WO 90 / 103430, and WO 87 / 00195, the entire contents of which are incorporated herein by reference, may be used as a culture medium for the host cells.

[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 phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), minor elements (defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions, e.g., temperature, pH, etc., will be those previously used with the host cell chosen for expression and will be apparent to those of skill in the art.

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

[0214] therapeutic use In one aspect, provided herein is a method of treating cancer in a subject by administering to a subject with cancer a pharmaceutical composition described herein.

[0215] In some embodiments, the methods disclosed herein are used in place of standard therapy. In certain embodiments, standard therapy is used in combination with any of the methods disclosed herein. Standard therapy for various types of cancer is 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, has published the NCCN Clinical Practice Guidelines in Oncology (NCCN GUIDELINES®), which provide detailed and up-to-date information on standard therapy treatments for a wide range of cancers. In some embodiments, the methods disclosed herein are used after standard therapy has failed.

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

[0217] In some embodiments, the cancer is driven by EGFR. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematopoietic 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, as detected, for example, by biopsy or fluorescence in situ hybridization.

[0220] In some embodiments, the cancer comprises cancer cells comprising a genomic alteration in the KRAS gene. In some embodiments, the alteration in the KRAS gene is a G12D substitution. In some embodiments, the alteration in the KRAS gene is a G13D alteration.

[0221] In some embodiments, 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 cancer, head cancer, and neck cancer.

[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, as determined by biopsy. In some embodiments, the SqCLC comprises cancer cells that contain genomic amplification of the EGFR gene, as 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 cancer. In some embodiments, the spinal 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 anaplastic thyroid cancer (ATC). In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric 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] Exemplary Dosing Regimen and Schedule In some embodiments, a 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 some embodiments, the fusion protein is about 50 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 300 mg to 2000 mg, 400 mg to 2000 mg, 500 mg to 2000 mg, 600 mg to 2000 mg, 700 mg to 2000 mg, 800 mg to 2000 mg, 9000 mg to 2000 mg, In some embodiments, the fusion protein is administered to a subject at a dose of about 200 mg to 2000 mg. In some embodiments, the fusion protein is administered to a subject at a dose of about 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, or 2000 mg. In some embodiments, the fusion protein is administered to a subject at a dose of about 64 mg, 240 mg, 800 mg, or 1600 mg. In some embodiments, the fusion protein is administered to a subject at a dose of about 64 mg. In some embodiments, the fusion protein is administered to a subject at a dose of about 240 mg. In some embodiments, the fusion protein is administered to the subject at a dose of about 800 mg. In some embodiments, the fusion protein is administered to the subject at a dose of about 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 aspect, a kit is provided herein that includes the liquid pharmaceutical composition described herein for therapeutic use.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 writing or recording material provided on or with the kit, or that accompanies the kit.Thus, the present disclosure provides a kit for treating a subject suffering from cancer, comprising (a) a dosage of the pharmaceutical composition described herein, and (b) instructions for use in the therapeutic method disclosed herein.In a specific embodiment for treating a human patient, the kit includes the liquid pharmaceutical composition described herein that includes BCA101.

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

[0232] [Example 1] Development of BCA101 formulation The goal of this study was to develop a bifunctional fusion protein formulation in which the two functional domains of the protein have different isoelectric points. This study utilized BCA101 as such a fusion protein. BCA101, as described herein, is a bifunctional fusion protein containing an anti-hEGFR antibody and the extracellular domain of hTGFβRII fused to the C-terminus of the anti-hEGFR antibody light chain. 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 that could accommodate a fusion protein containing two proteins with different functions, structures, and pIs. The formulation would need to maintain the physicochemical stability, functional, and biological potency 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 studies A pH screening study was conducted to determine the pH at which BCA101 was most stable. Ultrafiltration of BCA101 (approximately 35 mg / ml) was performed using tangential flow filtration (TFF) at pHs of 5.0, 5.5, 6.0, and 6.5, and the filtered product was formulated in 10 mM citrate phosphate buffer (10 mM), 0.02% w / v polysorbate 20, and 25 mg / ml BCA101 (Figure 2). The percentage of high molecular weight protein (HMWP) (Figure 3), percentage of protein monomer (Figure 4), and percentage of low molecular weight protein (LMWP) (Figure 5) were measured for the bulk tangential flow filtration composition (TFF) and the final drug product (FDP) by size exclusion chromatography. The stability of BCA101 in each of the pH formulations was further analyzed by differential scanning calorimetry (DSC). The DSC traces for each formulation are shown in FIG. 6, and a summary of the data at each pH is shown in FIG. 7 and Table 5.

[0234] [Table 5]

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

[0236] Buffer screening studies 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 selected pHs of 6.0 and 6.5. Drug products were 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 BCA101, 0.02% w / v polysorbate 20, and 10 mM test buffer (citric acid, succinic acid, histidine, and citrate phosphate) (Figure 11). The stability of BCA101 was measured by DSC for each formulation: citrate buffer (Figures 12A-12B), succinate buffer (Figures 13A-13B), histidine buffer (Figures 14A-14B), and citrate phosphate buffer (Figures 15A-15B). A summary 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 data above, citrate phosphate (pH 6.0) and succinate (pH 6.5) were selected for further development.

[0238] [Table 6]

[0239] Tonicity modifying agent screening Tonicity modifiers were screened for each of the selected buffers and pHs. Each test formulation contained 25 mg / ml BCA101, 0.02% w / w, 5.0% w / v tonicity modifier (sucrose or trehalose), and 10 mM buffer (citrate phosphate (pH 6.0) or succinate (pH 6.5)) according to Table 7 below (Figure 17).

[0240] [Table 7]

[0241] Stress Stability Studies Stress stability testing was performed at 40°C, with three freeze cycles in cryovials, and freeze-thaw studies with each freeze cycle being 48 hours at -80°C and -20°C, and each thaw cycle being 4 hours at 25°C in an incubator. The results of the freeze-thaw and stress stability studies are summarized in Table 8. No changes in pH, osmolality, or protein concentration were observed in any of the test formulations during either the freeze / thaw or stress studies.

[0242] [Table 8]

[0243] The osmolality of sucrose formulations was evaluated (Table 9). 5% w / v resulted in osmolality values ​​ranging from 170 to 240 mOsmol / kg. Sucrose concentrations were further optimized based on achieving a target osmolality value of 300 mOsmol / kg. The osmolality of BCA101 formulations with 0.02% w / v polysorbate-20 and 10 mM citrate phosphate buffer in the absence of sucrose was in the range of 30 to 35 mOsmol / kg. A sucrose concentration of 8.0% w / v is expected to achieve 300 mOsmol / kg for the BCA101 drug product.

[0244] [Table 9]

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

[0246] [Table 10]

[0247] [Table 11]

[0248] [Table 12]

[0249] The clarity and turbidity of BCA101 formulations containing 25 mg / ml BCA101, 0.02% w / v polysorbate 20, 8.0% w / v sucrose, and 10 mM citrate phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0) were evaluated in comparison with a pharmacopoeia standard (formazin suspension, Ph.Eu.2.2.1) (FIG. 20). The standard solution reference is provided in Table 13 below. The NTU values ​​of BCA101 samples are presented in FIG. 21. The assay was performed in accordance with Ph.Eur.2.2.1, the entire contents of which are incorporated herein by reference.

[0250] [Table 13]

[0251] Long-term stability testing The stability of BCA101 in a formulation containing 25 mg / ml BCA101, 0.02% w / v polysorbate 20, 8.0% w / v sucrose, and 10 mM citrate phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0) was evaluated for toxicology study batches at 2-20°C for the drug substance (DS) in 5 mL Celsius bags (Figures 22 and 25) and at 2-8°C for the drug product (DP) in glass vials (Figures 23 and 26). The pH, osmolality, protein concentration, and functionality of both arms of the BCA101 fusion protein (via a bifunctional ELISA measuring the ability to bind hEGFR and hTGFβ) of the drug substance (Figure 22) and drug product (Figure 23) were assessed over a 24-month period, with data points taken 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 from the long-term stability study between the drug substance and drug product is shown in Figure 24. Briefly, to perform the bifunctional ELISA, 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 will give a signal only if both arms are intact.

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

[0253] A diagrammatic representation of the exemplary formulation process described in the above examples is shown in FIG.

[0254] [Example 2] Long-term storage stability of BCA101 drug substance (DS) The objective of this study was to evaluate the long-term storage stability of BCA101 drug substance (DS) at -20 ± 5°C over a period of 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)), each containing 25 mg / ml BCA101, 0.02% w / v polysorbate 20, 8.0% w / v sucrose, and 10 mM citrate phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0) stored at -20 ± 5°C in either Flexboy bags (BL.14.0901 / R / 17 / 021 F DS) or Celsius FFT or Celsius Pak bags (BS17006883), were evaluated. pH, osmolality, protein concentration, % monomer, % high molecular weight protein (HMWP), % low molecular weight protein (LMWP), functionality of both arms of the BCA101 fusion protein (measured by bifunctional ELISA for their ability to bind 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 contaminating microbial counts were assessed over a 24-month period, with data points generally obtained at baseline, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months (except as noted in Tables 14 and 15 below). Tables 14 and 15 provide summaries of stability data for both batches of BCA101 DS. Descriptions and trend data for each of the individual stability studies 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°C, and the individual stability studies are described in more detail below.

[0256] [Table 14]

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

[0258] [Table 15-1]

[0259] [Table 15-2]

[0260] Visual observation Visual observation is a qualitative assessment of the description, clarity, and color of the manufactured drug substance. Based on manufacturing data, the following specifications were set for BCA101 DS: description: absence of intervening foreign particles set to "clear, cloudy liquid, free of foreign material," clarity: "cloudiness not exceeding Pharmacopoeia Reference Standard III (NTU value NMT 18 NTU)," and color: "not more strongly colored than Pharmacopoeia Reference Standard BY4." BS17006883 BCA101 DS batches were tested for visual observation and shown to meet each of the specifications for up to 24 months from the date of manufacture (see Table 15). BL.14.0901 / R / 17 / 021 F DS BCA101 DS batches were not tested for visual observation.

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

[0262] Osmolality Osmolality is one of the parameters that parenteral formulations must control to approximate that of plasma to avoid adverse reactions associated with the injection of hypo- or hypertonic DS during administration. The isosmolality of BCA101 is primarily achieved using sucrose in the formulation, and the osmolality specification for BCA101 DS was set at 270–330 mOsmol / kg. Any changes in sucrose concentration during the stability period may result in changes in osmolality, indicating an impact on the stability of the substance. As shown in Figure 30 and Tables 14 and 15, the osmolality of the BL.14.0901 / R / 17 / 021 F DS BCA101 DS batch was within the specification range for up to 24 months, and the BS17006883 BCA101 DS batch was also measured at 6 and 9 months and determined to be within the specification range. The osmolality of BS17006883 BCA101 DS batches has not been tested beyond the 9 month time point.

[0263] Protein concentration Protein concentration provides information about the amount of DS in a sample. Based on development data, the BCA101 DS protein concentration limit 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 specification for up to 24 months from the date of manufacture.

[0264] Contaminant Count and Bacterial Endotoxin Test (BET) The acceptance criteria for BET and microbial count for BCA101 DS were set at 0.25 EU / mg or less (NMT) and 10 CFU / 100 mL or less (NMT), respectively. As shown in Table 15, BS17006883 BCA101 DS batches met both BET and microbial count specifications for up to 24 months from the date of manufacture. BL.14.0901 / R / 17 / 021 F DS BCA101 DS batches were not tested for microbial count or BET.

[0265] Purity 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 Tables 14 and 15, SEC-HPLC purity results met specification limits for both BCA101 DS batches up to 24 months from the date of manufacture.

[0266] Purity by RP-HPLC RP-HPLC provides purity information for hydrophobic variants. The acceptance criteria for BCA101 DS were set as follows: NLT 70.0% for total main peak, NMT 24% for total post-peak, and NMT 6% for total pre-peak. As shown in Figure 35 (total pre-peak), Figure 36 (total post-peak), and Figure 37 (main peak %), and Tables 14 and 15, RP-HPLC purity results met the specification limits for BS17006883 BCA101 DS batches up to 24 months from the date of manufacture. BL.14.0901 / R / 17 / 021 F DS BCA101 DS batches were not tested by RP-HPLC.

[0267] Bifunctional ELISA A bifunctional ELISA was used to determine the simultaneous binding potency of BCA101 to the epidermal growth factor receptor (EGFR) and TGFβ1 ligand. 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 would yield a signal only if both antigen-binding arms of BCA101 (binding to EGFR and binding to TGFβ1 ligand) were intact. The assay acceptance criterion was set at a mean relative potency of 0.80 to 1.25 compared to the reference standard.

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

[0269] Inhibition of proliferation (IOP) assay An inhibition of proliferation (IOP) assay was used to determine the ability of BCA101 DS to inhibit FaDu cancer cell growth by binding to its target EGFR. This assay provides a method for determining the number of viable cells in culture by quantifying the amount of ATP present. Readout was based on luminescence from luciferin monooxygenation catalyzed by luciferase in the presence of Mg2+ and ATP released by viable cells. Assay acceptance criteria were set at a mean relative potency of 0.80 to 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 specification 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 presented above demonstrate that multiple batches of BCA101 drug substance stored at -20 ± 5°C for up to 24 months from the date of manufacture met specifications for various comprehensive critical quality attributes (release parameters). Based on stability trend analysis, no significant changes were observed in pH, osmolality, SEC-HPLC, protein content / concentration, RP-HPLC, or functionality. Data obtained from R&D (BL.14.0901 / R / 17 / 021 F DS) and developmental GMP batches (BS17006883) demonstrate the physicochemical and functional stability of the BCA101 drug substance formulation for up to 24 months from the date of manufacture 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 Product (DP) The objective of this study was to evaluate the long-term storage stability of BCA101 drug product (DP) stored at 5 ± 3°C for 24 months. Two different batches of BCA101 DP (BL.14.0901 / R / 17 / 021 F DS (R&D batch) and BS18002245 (GMP development batch)) containing 25 mg / ml BCA101, 0.02% w / v polysorbate 20, 8.0% w / v sucrose, and 10 mM citrate phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0) stored in 10R USP Type I clear glass vials at 5 ± 3°C were evaluated. pH, osmolality, protein concentration, % monomer, % 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 ability to bind hEGFR and hTGFβ), purity (by SEC-HPLC and RP-HPLC), inhibition of EGFR-expressing cell proliferation, visual interpretation, clarity, color, harvest volume, seal integrity, subvisible particulate matter, bacterial endotoxin, and sterility were assessed over a 24-month period, with data points generally obtained at baseline, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months (except as noted in Tables 16 and 17). Tables 16 and 17 provide summaries of stability data for the first and second batches of BCA101 DP. Descriptions and trend data for each of the individual stability studies are provided below and in Figures 40-51.

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

[0274] [Table 16]

[0275] Table 17 below provides a summary of long term stability data for BS18002245 BCA101 DS batches (GMP batches) stored at 5±3°C, and the individual stability studies are described in further detail below.

[0276] [Table 17-1]

[0277] [Table 17-2]

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

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

[0280] Osmolality Osmolality is one of the parameters that parenteral formulations must control to approximate that of plasma to avoid adverse reactions associated with the injection of hypo- or hypertonic DP during administration. The isosmolality of BCA101 DP is primarily achieved using sucrose, and the osmolality specification was set at 270–330 mOsmol / kg. Any change in sucrose concentration during the stability period may result in a change in osmolality, indicating an impact on protein stability. As shown in Figure 41 and Table 16, the osmolality of the BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch remained within the specification range for up to 24 months from the submission / manufacturing date. BS18002245 BCA101 DP batches were not tested for osmolality.

[0281] Protein concentration The 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), both batches of BCA101 DS met specifications for up to 24 months from the date of fill / manufacture.

[0282] Particles visible to the naked eye The specifications for macroscopic particles were set as follows: USP <790> Macroscopic particles in injection and USP <l>Injection (constituent solution), "The injectable preparation shall be substantially free of macroscopic particles." BCA101 DP vials were manufactured in a controlled environment and tested for macroscopic particles prior to batch release. No macroscopic particles were observed at the time of batch release or over the course of the stability study (24 months) (no changes are expected as the vials were aseptically sealed). BS18002245 BCA101 DP batches met specifications for up to 24 months from the date of manufacture (see Table 17). BL.14.0901 / R / 17 / 021 F DP BCA101 DP batches were not tested for macroscopic particles.

[0283] Particulate matter invisible to the naked eye Pharmacopoeia limits (USP <788> There are two specifications defined based on the size of sub-visible particles based on BS18002245: Sub-visible particles (10μm and above): No more than 6000 particles (NMT) per container, and Sub-visible particles (25μm and above): No more than 600 particles (NMT) per container. BS18002245 BCA101 DP batches met the specifications for up to 24 months from the date of manufacture (see Table 17). BL.14.0901 / R / 17 / 021 F DP BCA101 DP batches were not tested for sub-visible particulate matter.

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

[0285] Collection capacity Due to the dead volume present in the vial and stopper that contacts the product during storage, it is not possible to completely remove 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 fill volume for BCA101 DP was set at no less than 10.0 ml (NLT). The draw volume was therefore set at 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. BL.14.0901 / R / 17 / 021 F DP BCA101 DP batch was not tested for draw volume.

[0286] Seal Integrity Test Container Closure Seal Integrity Testing (CCIT) complements the sterility test and is performed to ensure microbial integrity (sterility) during storage and transport through to the end of the DP shelf life. The acceptance criterion for CCIT was set as "all tested vials do not contain any traces of colored solution." BS18002245 BCA101 DP batches met specifications for 24 months from the date of manufacture (Table 17). BL.14.0901 / R / 17 / 021 F DP BCA101 DP batches were not tested by CCIT.

[0287] Purity 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: NLT 93.00% for monomer %, NMT 3.00% for HMWP %, and LMWP for reporting results. As shown in Figure 44 (HMWP%), Figure 45 (monomer%), and Figure 46 (LMWP%), SEC-HPLC purity results met the specification limits for both BCA101 DP batches up to 24 months from the date of fill / manufacture.

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

[0289] With the exception of 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 date of manufacture. 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 the acceptance criteria from subsequent time points up to 24 months. This behavior was not observed in other analytical studies, and the BS18002245 BCA101 DP batch stability trend for up to 24 months from the date of manufacture was within the specified limits.

[0290] Bifunctional ELISA A bifunctional ELISA was used to determine the efficacy of BCA101 in simultaneously binding to EGFR and TGFβ1 ligands. 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 would yield a signal only if both binding arms were intact. The assay acceptance criterion was set at a mean relative potency of 0.80 to 1.25 compared to the 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 was well within the assay acceptance criteria up to 24 months from the date of fill / manufacture, the last time point tested. Similarly, the BS18002245 BCA101 DP batch results were well within the assay acceptance criteria up to 24 months from the date of fill / manufacture, the last time point tested (see Figure 50 and Table 17).

[0292] Inhibition of proliferation (IOP) assay An inhibition of proliferation (IOP) assay was used to determine the ability of BCA101 DP to inhibit FaDu cancer cell growth by binding to its target EGFR. This assay provides a method for determining the number of viable cells in culture by quantifying the amount of ATP present. Readout was based on luminescence from luciferin monooxygenation catalyzed by luciferase in the presence of Mg2+ and ATP released by viable cells. Assay acceptance criteria were set at a mean relative potency of 0.80 to 1.25 compared to a reference standard.

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

[0294] conclusion The data presented above demonstrate that multiple BCA101 drug product batches stored at 5±3°C met acceptance criteria for up to 24 months from the fill / manufacture date. Based on stability trend analysis, no significant changes were observed in pH, osmolality, subvisible particle count per container, SEC-HPLC, protein content, RP-HPLC, or functionality. Data from R&D (BL.14.0901 / R / 17 / 021 F DP) and developmental GMP batches (BS18002245) demonstrate the physicochemical and functional stability of BCA101 DP for up to 24 months from the fill / manufacture date when stored at 5±3°C in USP Type 1 glass vials. The BS18002245 BCA101 DP batches further met BET and sterility specification limits for 24 months from the fill / manufacture date, 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 BCA101, 0.02 wt / vol% polysorbate 20, 8.0 wt / vol% sucrose, and 10 mM citrate-phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0); DS preclinical R&D batch and internal reference standard, and 2) GMP batch GF19000040 containing 26.60 mg / ml BCA101, 0.02% w / v polysorbate 20, 8.0% w / v sucrose, and 10 mM citrate phosphate (citric acid monohydrate 0.573 mg / ml, disodium hydrogen phosphate dihydrate 1.294 mg / mL) buffer (pH 6.0). Table 18 provides a summary of the analytical tools utilized to evaluate the physicochemical and biological quality attributes of BCA101 in this example.

[0296] [Table 18]

[0297] The data in this example demonstrates the equivalence of BCA101 GMP DS batch GF19000040 to the internal reference standard batch BL.14.0901 / R / 17 / 021 / F DS, demonstrating that both batches meet the established quality attribute specifications. A summary of the physicochemical and biological characterization performed in this example is provided below and in Table 19, with additional detailed descriptions of each of the analyses performed below.

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

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

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

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

[0302] Product related variants: Size Variants—The percentages of monomeric, HMWP, and LMWP species were comparable and within the quality attribute specifications for both GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches as analyzed by SEC-HPLC (Table 20). The relative percentages of fragments quantified using nrCE-SDS and rCE-SDS were also comparable and within the specifications for GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches (Table 21). Charge Variants—The charge variants obtained after integration of the iCE analysis were designated 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 comparable and within the quality attribute specifications (Table 22). The RP profiles showing the hydrophobic variants—main, total pre-peak, and total post-peak and the corresponding relative area percentages—were comparable between GF19000040 and BL.14.0901 / R / 17 / 021 / F DS batches and 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 due to the association of two or more molecules of a monomer. The primary analytical method for the separation and estimation of HMWP is size exclusion chromatography HPLC (SEC-HPLC), which was used here. As shown in Table 20, the SEC-HPLC profiles of the first batch of BCA101 DS (GF19000040) and the second batch BL.14.0901 / R / 17 / 021 / F DS (internal reference standard) were visually similar (chromatograms not shown), and the relative percentages of monomer, HMWP, and LMWP 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 specifications of NMT 3.0% for HMWP and 7.0% for LMWP, respectively (see Table 20).

[0306] [Table 20]

[0307] Purity of BCA101 DS by nrCE-SDS and rCE-SDS Capillary electrophoresis (CE) is an automated and instrumented version of traditional slab gel electrophoresis (SDS-PAGE) that utilizes narrow-bore (20-200 μm inner diameter) capillaries to perform highly efficient separations of both large and small molecules. These separations are facilitated by the use of high voltages, which can generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. CE-SDS (reduced and non-reduced) is used for quantitative analysis of therapeutic product purity and size-based heterogeneity. BCA101 DS batches GF19000040 and BL.14.0901 / R / 17 / 021 / F DS, both of which were analyzed using non-reduced and reduced CE-SDS to quantify product-associated fragments. The non-reduced (nr) and reduced (r) CE-SDS electropherograms showed similarities across the two batches (chromatograms not shown), both within the quality attribute specifications (Table 21). The main peaks in the nrCE-SDS were observed to be broad and bimodal, which may result from a) incomplete denaturation of the large protein and b) heterogeneity in glycoforms resulting from heterogeneity in the denatured state. nrCE-SDS analysis of the GF19000040 batch and the BL.14.0901 / R / 17 / 021 / F DS batch showed corrected area percentages of the main peaks as 94.3% and 94.4%, respectively. rCE-SDS analysis showed that the ratios of the sum of the most abundant species (Peak 1 + Peak 2 + Peak 3) in the GF19000040 batch and the IRS batch were 97.5% and 97.8%, respectively. Thus, the ratio of fragments was similar across the two batches tested and within 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 biotherapeutics. This technique utilizes the principle of protein charge separation in a pH gradient gel matrix under an applied electric current. Based on net charge, proteins migrate through the gel matrix until equilibrium is reached between pH units and the molecular isoelectric point (pI). The charge variant profiles of both BCA101 DS batches were evaluated using the iCE technique. Due to the heterogeneity of BCA101 caused by heavy sialylation, the charge variant profile exhibits multiple peaks with a lack of baseline resolution. 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 relative area under the curve for each cluster. As shown in Table 22, the two batches were observed to be comparable within method variability and within specification.

[0310] [Table 22]

[0311] Intact mass analysis Intact mass analysis 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, rapid quantification tool performed by applying a laser beam to an analyte 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 ionization spectrum yields singly charged ions. Based on the cluster size distribution of the mass spectrum, software determines the average molecular mass of the analyzed sample. The 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 the amino acid sequence is 178,105 Da. As shown in Table 23, the observed molecular mass of BCA101 in the GF1900040 batch and the BL.14.0901 / R / 17 / 021 / F DS batch is approximately 192 kDa, which is higher than the theoretical mass of the molecule due to glycosylation. Due to the heterogeneity of N-linked glycosylation in BCA101, the mass spectrum shows a broad molecular weight distribution ranging from 180 kDa to 210 kDa.

[0313] [Table 23]

[0314] Peptide mass fingerprinting Reduced peptide mass fingerprinting (PMF) provides detailed information about proteins, including the determination of primary sequence, N- and C-terminal sequences, and identification of sites and types of post-translational modifications. This approach involves subjecting molecules to specific enzymatic digestion procedures followed by MS and / or MS analysis. 2 The peptides are subjected to chromatographic separation prior to analysis. 2 This approach facilitates the assessment of primary structure in terms of linker confirmation and N- and C-terminal sequencing. N- and C-terminal sequencing are important data sets for confirming the start and end of a protein sequence of interest. PMF analysis is widely used in the pharmaceutical industry to generate unique fingerprints of molecules of interest and aid in determining complete protein sequence coverage. PMF profile overlays of 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 Lys-C, were used for 100% sequence coverage. The heavy and light chain-linker-TGFβRII ECD fragments generated by multiple enzyme digestion were determined, along with their theoretical and observed masses and their respective retention times.

[0315] As shown in Figure 54, the UV chromatograms of reduced alkylated tryptic peptide mass fingerprinting of the GF19000040 batch, along with the BL.14.0901 / R / 17 / 021 / FDS batch, are comparable to each other and within the specifications. 2 Using multiple enzymes (Glu-C, AspN, LysC), no free light chains were found, and it can therefore be concluded that the fusions are intact in both batches.

[0316] N- and C-terminal sequencing N- and C-terminal sequencing experiments were performed to confirm the start and end of the protein sequence of BCA101. PMF was analyzed by MS and MS 2 It is a powerful method to use the data to provide N- and C-terminal sequences and to compare experimental spectral data with in silico generated tryptic digest LC and HC fragment masses. 2 The b and y daughter ion series of the N-terminal and C-terminal sequences of HC, LC, and linker obtained from the spectra are shown in Figures 55 to 59.

[0317] From the data shown in Figures 55-59, the N- and C-terminal sequences of the heavy and light chain TGFβRII were confirmed as follows: N-terminal HC:pyroQVQLK (SEQ ID NO: 35), C-terminal HC:SLSLSPG, N-terminal LC-TGFβRII:DILLTQSPVILSVSPGER (SEQ ID NO: 36), LC-linker-TGFβRII:GECGGGGSGGGGSGGGGSTIPPHVQK (SEQ ID NO: 37). The C-terminus of the TGFβRII ECD was identified by MS analysis due to its low intensity and high molecular weight. 2 was not selected for, however, the supporting charge states were visible (MS data provided above).

[0318] As shown in Figure 54, the UV chromatograms of the tryptic peptide maps of GF19000040 and BL.14.0901 / R / 17 / 021 / F DS corresponded 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 pyroQ in both analyzed batches. 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 show the presence of lysine, as "PGK." The N-terminus of the light chain is intact and does not show any modifications in the FmAb2 batch. The C-terminus of the light chain is fused to the TGFβRII ECD. The C-terminal sequence of the TGFβRII ECD was determined to be intact and consistent with the available theoretical sequence.

[0319] Circular dichroism (CD) Circular dichroism is a form of absorbance spectroscopy that measures the difference in absorption of right- and left-handed circularly polarized light by a substance. Protein secondary structure can be determined by CD spectroscopy in the "far-UV" spectral region (200-260 nm). At these wavelengths, the chromophores are peptide bonds, and signals arise when they are located in a regularly folded environment. α-helix, β-sheet, and random coil structures each yield CD spectra with characteristic shapes and magnitudes.

[0320] CD spectra of proteins in the "near-UV" spectral region (260-350 nm) can be sensitive to certain aspects of tertiary structure. At these wavelengths, the chromogens are aromatic amino acids and disulfide bonds, and the CD signals they produce are sensitive to the overall tertiary structure of the protein. Signals in the region from 250-270 nm are assigned to phenylalanine residues, signals from 270-290 nm are assigned to tyrosine, and signals from 280-300 nm are assigned to tryptophan. Tertiary structure is protein-specific and 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 exhibiting a minimum wavelength at 216.2 nm and the BCA101 DS GF19000040 batch exhibiting a minimum at 215.4 nm, indicating similar secondary and tertiary structures. A negative signature peak in the far-UV spectra of both batches was observed around 215 nm, indicating a characteristic β-sheet structure.

[0322] Non-reduced disulfide bridges The disulfide bonds between the heavy, light, and heavy-light chains of the BCA101 antibody framework determine its structure, stability, and biological function. The BCA101 antibody framework belongs to the IgG1 class and has 16 disulfide bonds, including 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, connecting the light and heavy chains. Cys 228 and Cys 231 in each heavy chain link to form two parallel interchain disulfide bonds between the two heavy chains. Four polypeptide chains (two heavy chains and two light chains) are connected by four interchain disulfide bonds to form a tetramer, which plays an important role in the backbone structure and function of antibodies. Disulfide scrambling or incomplete disulfide bond formation can result in loss of function. In addition, the TGFβRII-ECD domain of BCA101 is rich in Cys residues, which are linked to form six intrachain disulfide bridges, which play an important role in the structure and function of the receptor-binding domain. Disulfide linkages were analyzed to establish the presence of correct connections to ensure the function and quality of the drug.

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

[0324] [Table 24]

[0325] Among the disulfide linkages in the TGFβRII ECD, two of the tryptic peptides showed the presence of multiple disulfide bonds (peaks 10 and 11 in Table 25). To identify the linkages, further characterization studies were performed using multiple enzyme digestion and MS analysis. 2 Verification was performed using MS 2 was used to confirm the Cys258-Cys261 linkage.

[0326] [Table 25]

[0327] Table 26 shows the predicted masses of peptides from multiple enzyme digests along with their retention times (RT).

[0328] [Table 26]

[0329] As observed in Figure 62, the non-reduced PMF UV chromatogram of the BCA101 DS GF19000040 batch is comparable to the BCA101 BL.14.0901 / R / 17 / 021 / F DS batch and is within specification. The masses of disulfide-linked peptides after multi-enzyme proteolytic digestion for both batches are tabulated in Tables 25 and 26 and were found to be comparable across batches and to have 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 identified by MS analysis. 2 Furthermore, in one of the peptides generated by AspN digestion (...NCSIT....TVCH), Cys291-Cys308 was found to be identical to the GluC digest peptide MS. 2 The only other possible disulfide bond was that between Cys278 and Cys284, which was identified as the fourth disulfide linkage, since this had already been identified by

[14] . The masses of the corresponding peptides with disulfide bonds were confirmed to be equivalent across the two batches. Therefore, the disulfide-linked peptides in the TGFβRII ECD were analyzed by multiple enzyme digestion and MS analysis. 2 Confirmed by checking.

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

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

[0332] Table 27 below provides the relative abundance of the observed glycan species. Mass assignments of glycan species obtained using LC-ESI-MS were compared with MS 2 The 2-AA labeled N-glycan NP-HPLC chromatograms shown in Figure 63 and percent relative abundance (Table 27) of BCA101 DS GF19000040 batch and BCA101 DS BL.14.0901 / R / 17 / 021 / F DS batch are comparable and within the specification range.

[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 acid is of particular interest because it plays different roles in monoclonal antibody function.

[0335] The half-life of some glycoproteins can be enhanced by sialylation because sialic acid serves as a cap that hides the penultimate galactose residue that is recognized by the hepatic asialoglycoprotein receptor. Therefore, a better understanding and monitoring of sialylated glycans is needed with a view to obtaining safe and reliable monoclonal antibodies for various biotherapeutic applications.

[0336] In addition to affecting the biological and physicochemical properties of biopharmaceuticals, sialic acid moieties of protein therapeutics play a major role in serum half-life, as exposed galactose on glycoproteins is endocytosed by the hepatic asialogalactose receptor via receptor-mediated endocytosis. Release of sialic acid is via acid hydrolysis of monoclonal antibodies, followed by cleanup to remove the monoclonal antibodies and labeling of the sialic acid species with fluorescent tags via OPD. The tagged sample is injected onto a reverse-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 indicates that the peak corresponding to NGNA is absent in BCA101 DS based on the 25 and 300 pmol NGNA standard samples, which only shows a peak corresponding to NANA (based on the peak seen in the NANA standard sample). Thus, the data demonstrate the presence of NANA in BCA101, and that NGNA was not detected up to 20 pmol. As shown in Table 28, the average (n=4) sialic acid content in BCA101 DS (GF19000040) is estimated to be 8.8 moles per mole of protein, and that in BCA101 DS (BL.14.0901 / R / 17 / 021 / F DS) is estimated to be 12.2 moles per mole of protein. This difference in average sialic acid content is within the range of method variability and has little impact on biological function between batches, as shown in the functional assays presented below.

[0338] [Table 28]

[0339] Product Related Substances-RP-HPLC A reverse-phase HPLC (RP-HPLC) method is utilized to monitor product-associated hydrophobic variants, including post-translational modifications, oxidized proteins, clipped variants or fragments, N-terminal cyclization, etc. Proteins are separated on the column based on hydrophobicity, with less hydrophobic proteins eluting earlier than more highly hydrophobic variants.

[0340] Product-related hydrophobic variants in the BCA101 batches were classified into the main peak, peaks before the main peak grouped together as all pre-peaks, and peaks after the main peak grouped together as all post-peaks. The RP profiles and relative proportions of the main peak, pre-peak, and post-peak were observed to be similar across the 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 of which are within the specification of ≥70% (NLT). The total pre-peak percentages were observed to be 4.9% (GF19000140) and 4.8% (BL.14.0901 / R / 17 / 021 / F DS), both within the specification 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 of 24.0% or less (NMT).

[0341] [Table 29]

[0342] Inhibition of proliferation (IOP) BCA101 binds to the EGFR on FaDu cancer cells. Varying the drug concentration allows 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 culture by quantifying the amount of ATP present, which indicates the presence of metabolically active cells. The readout is based on luminescence from luciferin monooxygenation, catalyzed by luciferase in the presence of Mg2+, and the ATP released by viable cells.

[0343] The GF19000040 batch is 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 listed in Table 30 below.

[0344] As shown in Table 30, the GF19000040 batch exhibited an average relative potency of 0.99, which falls within the assay acceptance criteria of 0.8 to 1.25. The results indicate that GF19000040 and BL.14.0901 / R / 17 / 021 / F DS exhibit similar potencies for 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 moiety of BCA101 to its target protein, thereby determining that both moieties in BCA101 are simultaneously functional. FmAb2 has a TGFβRII ECD fused to an anti-EGFR monoclonal antibody at the C-terminus of the light chain. The TGFβRII ECD moiety primarily binds to TGFβ1, while the anti-EGFR moiety binds to EGFR. Individual target binding ELISAs can only independently determine the binding affinity of each moiety, and cannot determine bifunctionality.

[0347] The GF19000040 batches were evaluated for their bifunctional activity using BL.14.0901 / R / 17 / 021 / F DS as a standard. As shown in Table 31, GF19000040 exhibited a relative potency value of 0.93, which is well within the acceptance criteria of 0.8 to 1.25. The results further demonstrate that GF19000040 exhibits similar potency for bifunctional activity compared to BL.14.0901 / R / 17 / 021 / F DS.

[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 a surrogate readout at an early point in 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 the ADCC Bioassay Effector Cell Proliferation Model (Promega, catalog number G7102), which allows for cell banking and expansion. These cells are engineered Jurkat cells stably expressing the V158 (high affinity) variant of the FcγRIIIa receptor. Biological activity in ADCC is quantified through luciferase produced as a result of NFAT pathway activation. Luciferase activity in effector cells is quantified with a luminescent readout.

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

[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, a HEK293 cell line is engineered 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. TGFβ protein binds to a receptor on the cell surface, initiating a signaling cascade that leads to 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 a SMAD-binding element (SBE) in the nucleus, leading to the transcription and expression of TGFβ SMAD-responsive genes. 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, the GF19000040 batch was evaluated using BL.14.0901 / R / 17 / 021 / F DS as the standard. The GF19000040 batch exhibited a relative potency value of 1.10, which is well within the acceptable range of 0.80 to 1.25, and also exhibited a CV% of less than 20. This further establishes that the GF19000040 batch is equivalent to the BL.14.0901 / R / 17 / 021 / F DS batch.

[0354] [Table 33]

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

[0356] All references (e.g., publications, or patents, or patent applications) cited in this specification are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent, or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0357] Other embodiments are within the scope of the following claims.< / l>

Claims

1. A pharmaceutical composition comprising: a. a fusion protein comprising a targeting moiety and an immunomodulatory moiety, (i) the targeting moiety specifically binds to human epidermal growth factor receptor (hEGFR), and (ii) the immunomodulatory moiety comprises an amino acid sequence of the extracellular domain of human transforming growth factor-beta receptor II (hTGFβRII); b. a buffer present at a concentration between 5 mM and 30 mM; c. a tonicity adjusting agent present in a concentration of 4% to 10% w / v; Including, The pharmaceutical composition has a pH of 5.5 to 7.

0. Pharmaceutical compositions.

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

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a powder pharmaceutical composition.

4. 2. The pharmaceutical composition of claim 1, wherein the buffer is a citrate phosphate buffer.

5. 2. The pharmaceutical composition of claim 1, wherein the buffer is present at a concentration of 5 mM to 15 mM.

6. The pharmaceutical composition according to claim 1 , wherein the buffer comprises 10 mM phosphate citrate.

7. The pharmaceutical composition of claim 1 , wherein the tonicity adjusting agent is a sugar.

8. The pharmaceutical composition of claim 1 , wherein the tonicity adjusting agent is sucrose.

9. 10. The pharmaceutical composition of claim 1, wherein the tonicity adjusting agent is present in a concentration of 5% to 8% weight / volume.

10. 2. The pharmaceutical composition of claim 1, wherein the tonicity adjusting agent is sucrose and is present in a concentration of 8% weight / volume.

11. The pharmaceutical composition of claim 1 further comprising a surfactant.

12. 12. The pharmaceutical composition of claim 11, wherein the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

13. 12. The pharmaceutical composition of claim 11, wherein the surfactant is polysorbate 80 and is present at a concentration of 0.02% weight / volume.

14. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition has a pH of 6.0 to 6.

5.

15. The 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 2-8°C; the concentration of the fusion protein in the pharmaceutical composition is substantially the same for at least 12 months, 18 months, or 24 months when stored at −20° C. or 2-8° C.; the concentration of said fusion protein in said 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 or 2-8°C for 12 months, 18 months, or 24 months; the pharmaceutical composition is stable to 1, 2, 3, 4, or 5 cycles of freezing and thawing; 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. or 2-8° C.; and / or the pharmaceutical composition contains 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% of the fusion protein in aggregate form; The pharmaceutical composition of claim 1 , comprising one or more of:

16. The pharmaceutical composition comprises: After storage at -20°C or 2-8°C for 12, 18, 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. Reduced Viscosity The pharmaceutical composition of claim 1, having at least one characteristic selected from the group consisting of:

17. 2. The pharmaceutical composition of claim 1, wherein the fusion protein is present at a concentration of 25-50 mg / ml.

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

19. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety that specifically binds to hEGFR comprises an antibody or a functional fragment or variant thereof.

20. The antibody, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; a. VH CDR1 comprises an amino acid sequence 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 at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:2; c. the VH CDR3 comprises 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 antibody, or functional fragment or functional variant thereof, that specifically binds to hEGFR comprises a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3; d. VL CDR1 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:4; e. VL CDR2 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; f. VL CDR3 comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:6; 20. The pharmaceutical composition of claim 19.

21. The antibody, or functional fragment or functional variant thereof, which specifically binds to hEGFR, VH comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; A VL comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8; 20. The pharmaceutical composition of claim 19, comprising:

22. The antibody, or functional fragment or functional variant thereof, which specifically binds to hEGFR, a heavy chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10; a light chain comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:11; 20. The pharmaceutical composition of claim 19, comprising:

23. 2. The pharmaceutical composition of claim 1, wherein 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.

24. The pharmaceutical composition of claim 1 , wherein the immunomodulatory moiety is indirectly fused to the targeting moiety via a peptide linker.

25. 25. The pharmaceutical composition of claim 24, wherein the linker comprises the amino acid sequence of SEQ ID NO:

24.

26. the targeting moiety is an antibody comprising a light chain and a heavy chain, and the immunomodulatory moiety is fused to the C-terminus of the heavy chain of the targeting moiety; or the immunomodulatory moiety is fused to the C-terminus of the light chain of the targeting moiety; The pharmaceutical composition of claim 1.

27. 2. The pharmaceutical composition of claim 1, wherein the targeting moiety is an antibody that specifically binds to hEGFR, comprising a heavy chain comprising 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 comprising 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 moiety comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23, and wherein the N-terminus of the immunomodulatory moiety is indirectly fused to the C-terminus of the light chain through a linker, wherein the linker comprises an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

24.

28. 2. The pharmaceutical composition of claim 1, wherein the fusion protein comprises an antibody comprising a heavy chain comprising 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 comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

29.

29. A pharmaceutical composition comprising: a. a fusion protein at a concentration of 25-50 mg / ml comprising a targeting moiety and an immunomodulatory moiety, the fusion protein comprising an antibody comprising a heavy chain comprising 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 comprising 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. 5 mM to 20 mM citrate phosphate buffer; c. 6% to 10% w / v sucrose; d. 0.01% to 0.05% weight / volume of Polysorbate 80; and Including, The pharmaceutical composition has a pH of 5.5 to 6.

5. Pharmaceutical compositions.

30. A pharmaceutical composition comprising: a. a fusion protein comprising a targeting moiety and an immunomodulatory moiety at a concentration of 25-50 mg / ml, the fusion protein comprising an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 29; b. 5 mM to 20 mM citrate phosphate buffer; c. 6% to 10% w / v sucrose; d. 0.01% to 0.05% weight / volume of Polysorbate 80; and Including, The pharmaceutical composition has a pH of 5.5 to 6.

5. Pharmaceutical compositions.

31. A pharmaceutical composition comprising: a. 50 mg / mL fusion protein comprising a targeting moiety and an immunomodulatory moiety, the fusion protein comprising an antibody comprising a heavy chain comprising 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 comprising 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% w / v sucrose; d. 0.02% weight / volume of Polysorbate 20; Including, The pharmaceutical composition has a pH of 6.0±0.

3. Pharmaceutical compositions.

32. A pharmaceutical composition comprising: a. 50 mg / mL fusion protein comprising a targeting moiety and an immunomodulatory moiety, the fusion protein comprising an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 29; b. 10 mM citrate phosphate buffer; c. 8% w / v sucrose; d. 0.02% weight / volume of Polysorbate 20; Including, The pharmaceutical composition has a pH of 6.0±0.

3. Pharmaceutical compositions.

33. 10. The pharmaceutical composition of claim 1 for use in a method for treating human cancer in a subject having cancer.

34. The pharmaceutical composition of claim 33, wherein the cancer is a solid tumor.

35. 34. The pharmaceutical composition of claim 33, 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 cancer, head cancer, and neck cancer.

36. 34. The pharmaceutical composition of claim 33, wherein the cancer is head and neck cancer.

37. 34. The pharmaceutical composition of claim 33, wherein the cancer is head and neck squamous cell carcinoma (HNSCC).

38. 34. The pharmaceutical composition of claim 33, wherein the cancer is anal cancer.

39. 34. The pharmaceutical composition of claim 33, wherein the cancer is squamous cell carcinoma of the anal canal (SCCAC).

40. A method for producing a pharmaceutical composition, comprising: a. culturing a mammalian cell, the genome of which is stably integrated with one or more nucleic acids encoding a fusion protein comprising a targeting moiety and an immunomodulatory moiety, in a cell culture medium such that the cell secretes the fusion protein into the cell culture medium, wherein (i) the targeting moiety specifically binds to hEGFR, and (ii) the immunomodulatory moiety comprises an amino acid sequence of the extracellular domain of hTGFβRII; b. purifying the fusion protein from the cell culture medium; c. preparing the pharmaceutical composition of claim 1; The method includes: